Detection kit, assay plate to be used therein, detection method, evaluation method, polyclonal antibody of frog vitellogenin and process for producing the same

ABSTRACT

On a surface of a well previously disposed on a plate, a primary antibody that recognizes vitellogenin is solid-phased, in the well a sample obtained from a test body exposed to an environment is injected to react, followed by injecting a secondary antibody that is labeled with an enzyme and recognizes the vitellogenin, further followed by injecting a chromogenic reagent to cause a coloring reaction and by measuring the stained amount, still further followed by calculating an amount of vitellogenin from the stained amount to evaluate an environment based on the amount of vitellogenin.

FIELD OF THE INVENTION

The present invention relates to a technical field that evaluates anenvironment with, for instance, frog vitellogenin. In addition, thepresent invention relates to, in particular, a detection kit of frogvitellogenin, a measurement plate, a method of detecting vitellogenin,an evaluation method and polyclonal antibodies to frog vitellogenin.

BACKGROUND OF THE INVENTION

Recently, influences of various chemical substances on living thingsincluding humans and ecosystems are clearly existed.

Among these, influences of endocrine disruptors that are generallycalled as environmental hormones, disrupt an endocrine system of aliving thing and disturb the homeostasis thereof are becoming serious.

It is considered that the endocrine disruptor (hereinafter, referred toalso as environmental hormone) generates actions similar to hormonesthat a living thing originally has, or disturbs the actions, and therebycauses the abnormality to the living thing. As a working point of theendocrine disruptor, various phases such as binding with a hormonereceptor, binding with a hormone (ligand), synthesis of hormones in aliving thing and hormone metabolism are pointed out; however, sinceaction mechanisms of the endocrine disruptors on living things arediversified, at present, the mechanism of the endocrine disruptingactions due to the endocrine disruptors are not yet clearly understood.

Influences of the endocrine disruptors on the living things have beenconfirmed mainly with the malformation and the behavior disorder as anindicator. However, when the malformation or the behavior disorder istaken as an indicator, these are quantified with difficulty and both thesensitivity and the accuracy thereof are low; accordingly, it is veryrisky and difficult to estimate the actions of the chemicals from themalformation or the behavior disorder. In this connection, a molecularmarker that can quantitatively evaluate, before the abnormality in thephenotype of living things such as the malformation or the behaviordisorder are generated, the actions of the chemicals or the environmenton living things is in demand.

As one of the molecular markers that are provided with suchcharacteristics, vitellogenin that are yolk protein precursors of anegg-laying animal are gathering attention. Vitellogenin is normallyactively synthesized in breeding times in a liver of a femaleindividual; on the contrary, the vitellogenin is not detected or presentoriginally in male blood. From these characteristics, vitellogenin isgathering attention as a marker that can evaluate with the sensitivityand the quantitativity the endocrine disrupting action of the chemicalsor environment.

In order to detect vitellogenin, there are descriptions in, forinstance, JP-A Nos. 2001-218582, 2001-122899, and 2000-125867, onmethods of detecting vitellogenin of fishes such as carps and Oryziaslatipes, and in actuality detection kits of vitellogenin is practicallyused.

DISCLOSURE OF INVENTION

However, in order to understand the influences of the chemicals on anentire ecosystem and cope with these, the influences of the chemicals onnot only fishes but also living things located on various nutritionstages and phyletic evolution stages have to be properly investigatedand evaluated.

Furthermore, a structure of the vitellogenin is very complicated andrather diversified depending on the species; accordingly, it is verydifficult to measure vitellogenin due to other species with an existingdetection kit that is constituted for a particular species.

The present invention was achieved in view of the above situations andintends to provide a technology that can detect vitellogenin ofamphibians, in particular, frogs with the quantitativity and sensitivityto precisely evaluate the chemicals and the environment.

The present inventors studied hard and achieved the above object bymeans shown below.

That is, a detection kit of frog vitellogenin according to the inventionincludes a measurement plate having a plate body that has a bottomedwell wherein a sample is injected and primary antibodies that aresolid-phased on a surface of the well and recognizes the frogvitellogenin; standard frog vitellogenin that are injected in the wellwhere the primary antibodies are solid-phased; and secondary antibodiesthat are injected in the well where the sample or standard is injectedto recognize the frog vitellogenin.

Furthermore, according to a separate viewpoint, a detection kitaccording to another aspect of the invention includes a plate body thathas a bottomed well wherein a sample is injected; primary antibodiesthat are solid-phased on a surface of the well and recognizes the frogvitellogenin; standard frog vitellogenin that are injected in the wellwhere the primary antibodies are solid-phased; and secondary antibodiesthat are injected in the well where the sample or the standard isinjected to recognize the frog vitellogenin.

Here, the environment means the chemicals present in the environment orthe environment that is polluted with the chemicals.

Furthermore, the sample here is a blood plasma or blood serum, a tissueand a cell of a frog. Examples of the frog that can be used in theinvention include Rana japonica, Rana nigoromaculata, Rana rugosa,Microhyla ornate, Bombina bombina, Xenopuas laevis, and Xenopustropicalis. Among these, when the Xenopus laevis is used, irrespectiveof seasons, a large amount of eggs, resultantly, adults can be obtainedand individuals can be easily maintained. Accordingly, it can bepreferably used.

In the invention, according to the configuration as mentioned above, thefrog vitellogenin can be speedily and easily detected.

In the invention, in particular, primary antibodies that recognize thefrog vitellogenin is solid-phased on a surface of a well of a plate;accordingly, according to, for instance, sandwich enzyme-linkedimmunosorbent assay (ELISA) involving a detection method according tothe invention described below, vitellogenin can be detected.

An existing vitellogenin detection kit is for use in fishes alone;however, according to the invention, since vitellogenins of theamphibians including frogs can be measured with accuracy, evaluation ofthe chemicals and so on in these species can be properly carried out.

Furthermore, frogs are egg-lying animals and accumulate a large amountof yolk proteins in an egg in order to secure nutrition in the course ofevolution. The vitellogenin is a precursor of the yolk protein andnormally synthesized in a liver of a female individual; namely, it isnot synthesized in a liver of a male individual. However, in the case ofa male individual exposed to estrogen and so on, vitellogenin that isnot originally synthesized are synthesized in a liver. Accordingly,vitellogenin, even when the endocrine disrupting action mechanism due tothe endocrine disruptors is not clear, are effective as a marker thatcan evaluate the endocrine disrupting properties in the environment withthe sensitivity.

Still furthermore, since a life circle of frogs covers an amphibianenvironment, frogs are characteristic in that they are exposed not onlyto chemicals in water sites such as rivers, lakes, and undergroundwaters or environment soils but also to chemicals in air. Accordingly,when influences of the chemicals such as the environmental hormones onwildlife are evaluated, the frogs can be advantageously used.

Furthermore, the sample is blood plasma or blood serum of a frog.

As a sample that can be used in the detection kit according to theinvention, samples due to blood plasma or blood serum or a certain kindof tissues or cells of a frog that was exposed to the wildlife or theenvironment for a definite period, or of a frog that was exposed to thechemicals in a laboratory can be used.

When these are used as samples, the detection precision can be improved.

In the plate, a plurality of wells can be preferably disposed andvarious known plates can be used. Owing to the presence of the pluralityof wells, various kinds of samples can be simultaneously processed,resulting in an improvement in the processing efficiency.

In addition, since antibodies are solid-phased on a surface of each ofthe plurality of wells, when the sample is dispensed, anantigen-antibody reaction can be readily caused, resulting in a rapidprocessing.

In the invention, the secondary antibody is covalently coupled with alabeling compound.

Here, the labeling compound means enzymes such as HRP (horseradishperoxidase), biotin and so on.

Thereby, when the enzyme-labeled secondary antibodies or the secondaryantibodies and the third enzyme-labeled detection compounds thatrecognize the secondary antibodies are injected to the plate followed byreacting with a chromogenic reagent, the labeling enzymes develop acolor, and when an amount of stained is measured in terms of theabsorbance, an amount of vitellogenin can be quantified.

The primary antibodies are adsorbed and solid-phased on a surface of thewell and blocked by a blocking reagent.

Thereby, the solid-phased primary antibodies are assuredly fixed,resulting in an improvement in the reliability of detection results.

Furthermore, a detection kit involving still another aspect of theinvention includes a first plate that has a bottomed well in which asample and antibodies that recognize frog vitellogenin and areconjugated with a labeling compound are injected and mixed; a secondplate body that has a bottomed well in which a mixture liquid of thesample and antibodies is injected; and standard frog vitellogenin thatare solid-phased as antigens on a surface of the well of the secondplate.

Since according to the configuration like this, the detection kitbecomes one that is particularly suitable for an competitive methodamong enzyme-linked immunosorbent assay, the processing can beefficiently carried out according to the method and proper results canbe obtained.

Furthermore, the sample is preferable to be blood plasma or blood serumof a frog. Thereby, vitellogenin can be efficiently detected.

Still furthermore, the antigens are adsorbed and solid-phased on asurface of the well and blocked by a blocking reagent.

Thereby, nonspecific antibodies are excluded from binding; accordingly,the reliability of evaluation also can be improved.

The measurement plate according to the invention includes a plate bodyhaving bottomed wells wherein a sample is injected; and primaryantibodies that are solid-phased on a surface of the well and recognizethe frog vitellogenin.

According to the configuration like this, when it is used as ameasurement plate of a detection kit involving a sandwich method, thefrog vitellogenin can be efficiently detected.

That is, since antibodies are solid-phased beforehand on a surface ofthe plate, only by dispensing a sample there, speedily, anantigen-antibody reaction can be obtained. Furthermore, since there isno need of antibodies being solid-phased every time when the process isapplied, results can be inhibited from scattering.

Furthermore, a measurement plate involving another aspect according tothe invention includes a plate body that has bottomed wells in each ofwhich a mixture of a sample and antibodies that recognize frogvitellogenin and are labeled with a labeling compound is injected; andfrog vitellogenin that are solid-phased as antigens on a surface of eachof the wells of the plate.

According to the configuration like this, since in this case antigensare solid-phased on a surface of the plate, it can be used as ameasurement plate of a detection kit involving competitive binding assayantagonism, thereby frog vitellogenin can be efficiently detected.

The detection method according to the invention detects the frogvitellogenin with the above mentioned detection kit.

According to the configuration like this, since a detection kitexcellent in the sensitivity can be used, the reliability in thedetection can be improved.

Furthermore, a detection method of the frog vitellogenin involvinganother aspect includes reacting a sample and antibodies that recognizevitellogenin contained in the sample; and reacting a complex of thevitellogenin contained in the sample and the antibody with a secondaryantibody that is labeled with a labeling compound and recognizes thevitellogenin.

According to the configuration like this, since vitellogenin is detectedby means the enzyme-linked immunosorbent assay, the detection accuracycan be improved and a processing time can be shortened. In particular,the detection method according to the invention is suitable for thesandwich method and can improve the detection accuracy of vitellogenin.

Furthermore, reacting the secondary antibody in the complex with achromogenic reagent and measuring an amount of vitellogenin in thesample based on the coloring reaction is further included.

Thereby, the labeled vitellogenin can be assuredly quantified accordingto the sandwich method.

Furthermore, still another aspect involving the detection method of thefrog vitellogenin according to the invention includes reacting a sampleand antibodies that are labeled with an enzyme and recognizevitellogenin contained in the sample to obtain a complex; andcompetitively reacting the complex and the vitellogenin.

The detection method involving the aspect is an competitive method, andaccording to the configuration like this, the detection sensitivity ofvitellogenin can be improved in accordance with a shape of the sample.

Furthermore, the detection method further includes reacting a reactionproduct obtained owing to the competitive reaction and a chromogenicreagent and measuring an amount of vitellogenin in the sample based on acoloring reaction.

Thereby, vitellogenin detected owing to the competitive reaction can beassuredly quantified.

Thus, the detection method according to the invention is a so-calledenzyme-linked immunosorbent assay (ELISA method) and can be preferablyapplied to both the sandwich method and the competitive method.

Thereby, even when various samples are used, the enzyme-linkedimmunosorbent assay excellent in the sensitivity in accordance with asample shape can be used.

Furthermore, an antibody that is used in the detection method may be anyone of a polyclonal antibody and a monoclonal antibody.

Still furthermore, the sample is preferably blood plasma or blood serumof a frog. Thereby, the detection sensitivity can be improved.

An evaluation method according to the invention includes reacting asample and antibodies that recognize vitellogenin contained in thesample; reacting secondary antibodies that are labeled with a labelingcompound and recognize the vitellogenin to complexes of vitellogenincontained in the sample and the antibodies; reacting labeling enzymes inthe secondary antibodies bonded to the complexes and a chromogenicreagent to measure a stained amount; and calculating an amount ofvitellogenin from the stained amount and evaluating an environment basedon the amount of vitellogenin.

According to the configuration like this, based on the sandwich method,accurate environmental evaluation can be carried out.

Furthermore, the sample is preferably blood plasma or blood serum of afrog.

Still furthermore, an evaluation method of another aspect involving theinvention includes reacting a sample and antibodies that are labeledwith a labeling compound and recognize vitellogenin contained in thesample to obtain complexes; causing the complexes and vitellogenin tocompetitively react; and reacting reaction products obtained owing tothe competitive reaction and a chromogenic reagent, calculating anamount of vitellogenins based on the coloring reaction to evaluate anenvironment based on the amount of vitellogenins.

Also in the aspect, the sample is preferably blood plasma or blood serumof a frog.

According to the configuration like this, based on the competitivemethod, accurate environmental evaluation can be performed.

Furthermore, as mentioned above, in the evaluation method according tothe invention, both the sandwich method and the competitive method canbe appropriately applied; accordingly, in accordance with various sampleshapes, evaluations can be performed.

Polyclonal antibodies of frog vitellogenin according to the inventioncan be obtained in such a manner that a mammal is immunized with frogvitellogenin as an antigen, antiserum are sampled from the immunizedmammal, and IgG are isolated from the antiserum.

When thus obtained antibodies are used, the frog vitellogenin can bedetected specifically and with sensitivity, resulting in properlyevaluating the environment.

In a manufacturing method according to the invention of polyclonalantibodies, IgG obtained from antiserum that are sampled from a mammalimmunized with frog vitellogenin as an antigen are purified by use of anaffinity column, and thereby polyclonal antibodies can be obtained.

Furthermore, the affinity column is bonded with male frog serumproteins.

Still furthermore, the affinity column is bonded with frog vitellogenin.

When thus configured, since binding specificity of the antibodies asthat can be improved, thereby the accuracy in the detection andevaluation method can be improved as well.

An evaluation method involving another aspect of the invention includescultivating hepatocytes of an amphibian; administering a sample chemicalto the hepatocytes; and detecting a response to a sample chemical of thecultivated hepatocytes.

Here, as the hepatocytes used here, ones from amphibians includingadults and larvae can be used.

Furthermore, the evaluation samples here indicate various kinds ofchemicals, and environmental samples such as river water, factorywastewater, water processed at sewage plants or extracted componentsfrom soil.

According to the configuration like this, with hepatocytes, to a lot ofsamples, the response of the hepatocytes can be directly detectedeconomically, speedily, conveniently, and without being influenced byhormones in living things, and a strict exposure condition also can beset.

A detection method involving still another aspect of the inventiondetects vitellogenin by use of the abovementioned detection kit.

Here, the response mainly indicates induction of vitellogenin synthesis.However, other than this, with induction of transferrin, albumin, and soon as a marker, actions of the chemicals belonging to otherenvironmental hormones can be variously evaluated.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view showing an entirety of a detection kitaccording to the present invention.

FIG. 2 is a schematic view of an entirety of a measurement plate of adetection kit according to the invention.

FIG. 3 is a partial sectional view of a well of a measurement plateinvolving a sandwich method of a detection kit according to theinvention.

FIG. 4 is a partial sectional view of a well of a measurement plateinvolving an competitive method of a detection kit according to theinvention.

FIG. 5 is a process chart explaining an competitive method in adetection method according to the invention.

FIG. 6 is a process chart explaining a sandwich method in a detectionmethod according to the invention.

FIGS. 7A and 7B are images showing the specificity of a vitellogeninantibody according to the invention.

FIGS. 8A and 8B are graphs showing the sensitivity in detectingvitellogenin in detection methods according to the invention.

FIG. 9 shows a graph and an image showing detection of vitellogenin in awestern blotting as a comparative example.

FIG. 10 shows graphs showing induction of vitellogenin synthesis inexposure in water according to an environmental evaluation methodaccording to the invention.

FIG. 11 shows graphs showing induction of vitellogenin synthesis indirect injection according to an environmental evaluation methodaccording to the invention.

FIG. 12 is a graph showing induction of vitellogenin synthesis in whichcultivated hepatocytes are used.

FIG. 13 is an explanatory diagram of a method for afinity-purifiedpolyclonal antibodies.

FIG. 14 shows an isolating purification chart pattern of VTGs owing toanion exchange chromatography (upper one) and a photographSDS-PAGE/CBB-staining images of a fraction solution (lower one).

FIG. 15 shows examples of concentration quantification of a VTGstandard.

FIG. 16 is a diagram showing a gel-stained pattern of a VTG standard.

FIG. 17 shows a diagram showing results of confirmation ofconcentrations of VTG standards according to an ELISA method.

FIG. 18 is a diagram showing a rise in specific antibody titers afteraffinity purification.

FIG. 19 is a diagram showing comparison of specific antibody titersbetween affinity-purified polyclonal antibodies of differentimmunization lots.

FIGS. 20A and 20B are diagrams showing the specificity of antibodiesagainst vitellogenin, 20A showing a CBB-stained pattern of a bloodplasmaample, 20B showing western blotting.

FIG. 21 is a diagram showing results of study on conditions formaximizing quantity of concentrations of solid-phased antibodies.

FIG. 22 is a diagram showing results of study on conditions formaximizing detection level by use of HRP labeled antibodies.

FIGS. 23A through 23D are diagrams showing addition/recovery rateexperiments and studies on BSA addition for improving the recovery rate,23A showing a case where the BSA is not included, 23B showing a casewhere 0.5 percent of BSA is included, 23C showing a case where 1 percentof BSA is included, 23D showing calibration curves.

FIG. 24 is a diagram showing the linear regression curves in the sampledilution experiments after a sample dilution liquid is optimized.

FIG. 25A is a diagram showing a situation where a frog is wrapped withtissue paper.

FIG. 25B is a diagram showing a situation where a frog is stabbed at aflank with a needle.

FIG. 25C is a diagram showing a situation where blood is flowing out inball from a flank of a frog.

FIG. 25D is a diagram showing a situation where blood is being sampledfrom a frog.

FIG. 26 is a diagram showing an ELISA KIT calibration curve.

FIG. 27 is a diagram showing numerical values characterizing the ELISAKIT calibration curve.

FIG. 28 is a diagram showing an ELISA calibration curve obtained by useof biotinylated antibodies.

FIGS. 29A and 29B are diagrams showing the cross-reactivity of antibodyto VTGs of other species of frogs, 29A showing a case of ELISA, 29Bshowing a case of SDS-PAGE/CBB staining.

FIG. 30 is a diagram showing VTG synthesis induction in hepatocyte inprimary culture.

FIG. 31 is a diagram showing a antagonistic effect on VTG synthesis inhepatocyte in primary culture.

FIG. 32 is a diagram showing a vitellogenin (VTG) calibration curve(part 1).

FIG. 33 is a diagram showing a vitellogenin recovery rate curve (part1).

FIG. 34 is a diagram showing a vitellogenin (VTG) calibration curve(part 2).

FIG. 35 is a diagram showing a linear decline of the response due to thevitellogenin dilution-dependent linear regression (part 2).

FIG. 36 is an exploded diagram of an immunochromatography deviceinvolving application examples according to the invention.

BEST MODE FOR CARRYING OUT THE INVENTION

In what follows, embodiments according to the present invention will beexplained with reference to the drawings.

Firstly, a detection kit will be explained. The detection kit is onethat can accurately detect frog vitellogenin by enzyme-linkedimmunosorbent assay (ELISA) and can cope with both a sandwich method andan competitive method of the ELISA.

Firstly, a detection kit that detects frog vitellogenin by means of asandwich ELISA will be explained based on the drawings. FIG. 1 is adiagram schematically showing a configuration of the whole of avitellogenin detection kit involving the present embodiment.

Here, a vitellogenin detection kit 1 includes a plate 10 having bottomedwells 101 in each of which a primary antibody 12 that recognizevitellogenin is solid-phased and therein a sample is injected; referencefrog vitellogenin 11 as a specimen; and secondary antibodies 13 labeledwith an enzyme or a biotin.

Furthermore, other than these, a test body dilution liquid 14 fordiluting a sample, an antibody dilution liquid 15 for dilutingantibodies, a base solution and a chromogenic reagent 16 for developingcolor due to labels of the secondary antibodies 13, a buffer solution 17for stabilizing a liquid in process, a reaction stop liquid 18 forsuppressing an excessive reaction or a cleaning liquid 19 for washingthe plate at a predetermined time, and a reagent normally used in theELISA method.

In the detection kit 1, according to the configuration like this, intothe wells 101 of the plate 10, samples prepared from frogs exposed to anenvironment are injected, an antigen-antibody reaction betweenvitellogenin contained in the sample or the specimen 11 and the primaryantibodies 12 or secondary antibodies 13 is caused, followed bydetecting it based on the ELISA method, and thereby vitellogenin can bedetected with high sensitivity.

Here, as samples that are used in the detection kit 1, samples derivedfrom body fluids or tissues or cells of a frog that is exposed to thewildlife or environment for a definite period, or a frog that is exposedto the chemicals in a laboratory can be used. As will be mentionedbelow, blood plasmas, a homogenate of liver, or hepatocytes in primaryculture of a frog can be preferably used. Vitellogenin is synthesized ina liver and transferred by blood; accordingly, with these as samples,vitellogenin can be detected with accuracy.

Furthermore, the sample is preferable to be frog blood plasmas or bloodserum. Still furthermore, the sample is preferable to be media ofcultured hepatocytes.

In the case of the sample being the tissue, a liver homogenate can bepreferably used, and in the case of it being the cell, (x laevis)hepatocytes in primary culture are preferable.

Thereby, the detection sensitivity of vitellogenin in a frog body can beimproved.

Furthermore, these samples, the primary antibodies, the secondaryantibodies and the specimen vitellogenin is diluted with theabovementioned buffer solution to process.

Still furthermore, as a label of the secondary antibody, horseradishperoxidase (HRP) or biotin is preferable; however, various other knownlabels can be used.

FIG. 2 is a schematic diagram of a plate 10 of the detection kit 1involving the embodiment.

As shown in FIG. 2, on the plate 10 of the detection kit 1, there isdisposed a plurality of wells 101 in which a sample is discharged. Here,12 plates on each of which, for instance, 8 wells are disposed in onerow are connected and thereby 96 wells are disposed, and in each of thewells 101 a series of processes is carried out. A partial sectional viewof the well is shown in FIG. 3.

On a surface of the well 101, by use of a sandwich method of detectionmethods described below, antibodies are solid-phased beforehand. In FIG.3, as an example, a state where primary antibodies 111 are solid-phasedis shown.

In the next place, a configuration of a detection kit that is used in ancompetitive method will be roughly explained. Reagents and so on thatare used as a kit are substantially similar to those of the sandwichmethod. However, as shown in FIG. 4, in the competitive method, in awell 201 of a plate 20, as an antigen, frog vitellogenin 211 aresolid-phased, in a separate plate (not shown in the drawing) a sampleand labeled antibodies are beforehand mixed, a mixture thereof isinjected in the plate 20 followed by measuring the reaction, and therebyvitellogenin can be detected.

Thus, when the detection kit involving the embodiment is used, even whena concentration of vitellogenin contained in the sample is low, it canbe detected with sensitivity and thereby more refined environmentalevaluation can be carried out. Furthermore, when antigens or antibodiesare solid-phased beforehand, the convenience also can be improved, theprocessing can be sped up and the reliability of obtained results can beimproved as well.

An existing kit for detecting vitellogenin can be applied only to thedetection of vitellogenin of Oryzias latipes or carps and cannot copewith higher animals having a somatic system higher than the fishes.However, according to the inventive kit, vitellogenin of the amphibians,frogs, can be detected, and thereby over-all environmental evaluationcan be performed.

In the next place, a detection method of frog vitellogenin will beexplained.

In a detection method involving the embodiment the abovementioneddetection kit is used, and, based on the enzyme immunoassay, that is,the ELISA method, vitellogenin is detected.

Now, in the ELISA method, two methods of a sandwich method and ancompetitive method are known. However, the detection method can copewith both.

In what follows, processes in the respective methods will be explainedwith reference to the drawings. FIG. 4 is a process chart for explainingsteps of the detection method according to the competitive method. FIG.5 is a process chart for explaining steps of the detection methodaccording to the sandwich method.

Competitive Method

Firstly, as solid-phased antigens, a preparation is solid-phased. Apreparation vitellogenin is diluted, followed by dispensing on amicro-plate, further followed by incubating (step 401), still furtherfollowed by immobilizing. After a predetermined time has passed, theplate is washed (step 402) and a blocking reagent is dispensed to applythe blocking on a surface of a well of the plate (step 403). Thereafter,the plate is washed to remove an excessive chemical (step 404).

In parallel with the immobilization, on a separate plate, HRP labeledantibodies and a sample (or antigens) derived from a frog exposed to anenvironment are mixed (step 4001), followed by incubating (step 4002).Thus obtained antigen-antibody complexes are dispensed on aantigen-solid-phased plate to cause an competitive reaction (step 405).Subsequently, the plate is washed (step 406) followed by injecting achromogenic substrate to cause a coloring reaction (step 407). When thereaction comes to completion, the absorbance is measured with amicroreader or the like (step 408) and from a value thereof an amount ofvitellogenin is calculated (step 409).

Sandwich Method

A diluted solution of primary antibodies is dispensed in each of wellsof a micro-plate followed by incubating for a predetermined period (step501). Thereafter, the plate is washed (step 502), a blocking reagent isdispensed to complete immobilization (step 503), followed by dispensinga sample derived from frogs in an environment or a preparationvitellogenin (diluted solution) to cause reacting (step 504).Thereafter, the plate is washed (step 505), followed by dispensingsecondary antibodies to react (step 506). Subsequently, the plate iswashed (step 507) followed by dispensing a chromogenic substrate tocause a coloring reaction (step 508). After the coloring reaction, theabsorbance is measured with a microreader or the like (step 509) andfrom a result thereof an amount of vitellogenin is calculated (step510).

As mentioned above, according to the detection method, two types ofdetection method can be used and various samples can be used.Furthermore, thereby, the detection sensitivity as well can be improved.

In the next place, an environmental evaluation method will be explained.

An environmental evaluation method involving the embodiment, by use ofthe abovementioned detection kit and the detection method, enables touse frog vitellogenin that has not been detected. Thereby, influences ofthe endocrine disruptors such as environmental hormones and variouskinds of chemicals in the environments can be evaluated.

As a vitellogenin detection kit that is put into practical use atpresent, there is only one that can cope only with the fishes such asOryzias latipes and carps. Accordingly, there is an inconvenience inthat a comprehensive environmental evaluation including living organismsother than the fishes cannot be carried out. However, according to theenvironmental evaluation method according to the invention, owing to theuse of frogs, there is an advantage in that based on an amount ofvitellogenin detected from a frog, the environmental evaluation can becomprehensively performed.

Now, as a method of exposing a frog that is used as a sample to anenvironment, methods such as addition of a target substance to breedingwater and direct injection to individuals can be used. Furthermore, bycapturing a frog in wildlife and obtaining a sample therefrom, thenatural environment as that can be easily and accurately evaluated;accordingly, evaluations of various environments can be performed.

In the environmental evaluation method involving the embodiment, samplesare prepared from each of a thus obtained test body and a test body bredin a reference area, an amount of vitellogenin in the test body ismeasured and compared with that of the test body in the reference area,and therefrom the endocrine disrupting action of the target substanceand the toxicity of the chemicals can be judged. That is, a male frog isbred in the presence of a chemical material (such as bisphenol A andphthalic acid ester) that is a target of evaluation, and an amount ofvitellogenins in blood plasma is measured with time and compared withthat of vitellogenin in blood serum obtained from a frog in thereference area. As a result, when an amount of vitellogenins obtainedfrom the amphibians in an experimental area is higher than that in thereference area, it can be judged that the chemical material that is atarget of evaluation causes the endocrine disrupting influences.Alternatively, after a male frog is bred in the coexistence of estrogenthat is a reference and a chemical substance that is a target ofevaluation, blood or the like is sampled, vitellogenin contained in thesample are reacted with antibodies according to the invention followedby measuring an amount of vitellogenins, and from the magnitudes ofobtained measurements the toxicity (such as the endocrine disruptinginfluences) of the chemical substance can be evaluated. That is, in thepresence of the chemical substance that is a target of evaluation, amale frog is bred, and an amount of vitellogenins in blood serum ismeasured with time and compared with that obtained a frog bred in thereference area. As a result, when an amount of vitellogenins obtainedfrom a frog in an experimental area is lower than that in the referencearea, it can be judged that the chemical substance that is a target ofevaluation causes the endocrine disrupting influences.

Furthermore, when a vitellogenin concentration when a frog is bred inthe presence of a certain concentration of estrogen and a vitellogeninconcentration when a frog is bred in the presence of a chemicalsubstance that is a target of evaluation are compared, the intensity ofthe endocrine disruption effect of the chemical substance that is atarget of evaluation can be evaluated relative to the intensity ofestrogen. That is, when an amount of vitellogenins when a frog is bredin the presence of a certain concentration (such as 1.0 ppm) of estrogenis same as that when a frog is bred in the presence of a certainconcentration (such as 0.1 ppm) of a chemical substance of whichintensity of the endocrine disruption effect is not known, the endocrinedisruption effect of the chemical substance can be judged as 10 timesstronger than estrogen.

Furthermore, according to the evaluation method, when an amount ofvitellogenins in a body liquid of a male frog living in a river or alake that is a target of evaluation is measured, a situation ofpollution of the environment owing to the chemical substance having theendocrine disruption effect can be evaluated.

In the next place, a polyclonal antibody of frog vitellogenin will beexplained.

A polyclonal antibody of the frog vitellogenin can be manufacturedaccording to any one of so far known methods (such as Sambrook, J etal., Molecular Cloning, Cold Spring Harbor Laboratory Press (1989)).However, an antibody manufactured according to a manufacturing method ofpolyclonal antibody described below is preferable. In what follows,based on the manufacturing method, the polyclonal antibody will bedescribed.

To a mammal such as a rat, a mouse and a rabbit, frog vitellogenin isadministered as an antigen to immunize. An amount of administration ofthe antigen per one animal is, when, for instance, an adjuvant is used,500 to 1000 μg. As the adjuvant, Freund's complete adjuvant (FCA),Freund's incomplete adjuvant (FIA) and aluminum hydroxide adjuvant canbe cited. The immunization is mainly performed by hypodermic injection.Furthermore, an interval of the immunization is not particularlyrestricted; however, it may be applied with an interval from severaldays to several weeks, and preferably after 3 weeks from a firstimmunization, 1 to 2 times at an interval of 2 weeks. After 5 to 20 daysfrom the last immunization day, preferably after from 7 to 14 days,antiserum are sampled. Subsequently, from the obtained antiserum, bymeans of ammonium sulfate fractionation and DEAE-Sephadex columnchromatography, IgG are obtained.

In order to obtain vitellogeninpecific antibodies, by use of anabsorption column covalently coupled with frog serum proteins,adsorption purification is carried out, and by use of a frogvitellogenin column affinity purification is carried out.

Thus, not only IgG are isolated from the antiserum but also by applyingthe affinity purification, an improvement in the specificity andsensitivity of the polyclonal antibody can be realized.

According to a manufacturing method of vitellogenin involving theembodiment, the induction of vitellogenin synthesis is carried out in afrog body, blood is sampled from the frog followed by sampling a serum,and the serum is fractionated and purified. As a method of isolating andpurifying a serum, centrifugal separation, gel-filtration columnchromatography and so on can be properly combined, and therebyvitellogenin can be efficiently and accurately manufactured.

EXAMPLES

In what follows, the present invention will be detailed with referenceto examples. However, it goes without saying that the invention is notrestricted thereto.

Example 1 Vitellogenin Assay with Xenopus laevis

(1) Equipment: protein purification system, micro-plate reader(manufactured by Tosoh Corporation)

(2) Materials: male adult Xenopus laevis 17β-estradiol: CAS No.:50-28-2, molecular formula, molecular weight: 272.4 Lot No.: 19C-0519(available from SIGMA)

anti-vitellogenin rabbit antiserum: one prepared in year of 1979 andpreserved at −80 degrees centigrade was used.

(3) Example of manufacture of vitellogenin antigen

In order to obtain vitellogenin, to a male adult Xenopus laevis, apropylene glycol solution containing 10 mg/ml of 17β-estradiol(hereinafter referred to as E2) was injected at a concentration of 30μg/g BW (Body Weight) and thereby the induction of vitellogeninsynthesis was carried out. Blood sampled after 8 days' breeding wascoagulated, followed by centrifuging at 15000 rpm for 5 min at 4 degreescentigrade, and thereby a supernatant (blood serum) was sampled. Every 1ml of the blood serum sample was isolated and purified by use of ananion exchange chromatography system (QAE-Sephadex, manufactured byBio-Rad Econosystem) and thereby a vitellogenin preparation wasobtained. With BSA (bovine serum albumin) as a reference protein, thevitellogenin preparation was quantified with a BCA reagent followed byrendering a 50 percent glycerol solution at 0.5 mg/ml further followedby preserving at −20 degrees centigrade. This was used as a standardvitellogenin solution in experiments below.

(4) Example of Manufacture of Vitellogenin Antibody

From 10 ml of anti-vitellogenin rabbit antiserum frozen and preserved at−80 degrees centigrade, according to the ammonium sulfate fractionation,IgG antibodies were recovered. In order to purify antibodies againstvitellogenin, the recovered IgG antibody solution was purified by use ofa Sepharose 4B adsorption column thereto blood serum proteins of anormal male Xenopus laevis are conjugated. Furthermore, by use of apurified vitellogenin-conjugated Sepharose 4B column, the affinitypurification was applied. In order to prepare labeled antibodies, to thepurified antibodies, according to a periodic acid oxidation method,horseradish peroxydaze (HRP) was covalently coupled (HRP labeledpolyclonal antibody).

Thus, two kinds of the adsorption-purified polyclonal antibodies and theaffinity-purified HRP-labeled polyclonal antibodies were used asantibody solutions in experiments below.

(5) Vitellogenin Detection Method Due to Western Blotting (ComparativeExample)

A gel of SDS-7.5 percent acrylamide was prepared, 0.05 μl of each ofblood serum of an adult male Xenopus laevis administered as mentionedabove with E2 by injection and normal male blood serum thereof and adilution sequence in the range of 7.5 to 750 ng of purified vitellogeninantibodies were electrophoresed, and isolated proteins in the gel wereblotted on a membrane according to a semi-dry blotting method. Animmunoreaction was performed with 1 μg/ml of vitellogenin polyclonalantibodies followed by reacting with HRP labeled anti-rabbit IgG goatantibodies, and vitellogenin were detected on an X-ray film by means ofa chemical fluorescence development method. Thereby, the specificity ofthe antibody and the detection sensitivity due to the western blottingwere verified.

(6) Vitellogenin Detection Method by ELISA

The vitellogenin detection due to the ELISA was carried out according totwo methods of a) sandwich method and b) competitive method, and with adilution sequence of a purified vitellogenin preparation, the detectionsensitivities were compared and studied.

The respective detection procedures were determined as follows afterpreliminary experiments.

Example 1a Competitive Method

1. Immobilization of Vitellogenin Antibody

Each of 50 μl of standard vitellogenin diluted at 5 μg/ml with PBS wasdispensed on a microplate followed by incubating at 37 degreescentigrade for 2 hr.

-   1. Plate washing (0.1 percent Tween 20-PBS)-   2. Blocking of plate (0.5 percent I-Block, 0.1 percent Tween 20-PBS)-   3. Competitive reaction of antigen and HRP-labeled polyclonal    antibody

On a separate plate, 30 μl of antigens and 30 μl of 1 μg/ml polyclonalantibodies (diluted with a blocking solution) labeled with HRP weremixed followed by incubating at room temperature for 1 hr.

-   4. Washing of solid-phased plate (0.1 percent Tween 20-PBS)-   5. Immune reaction to solid-phased antigen

Fifty micro-litters of a blend solution of 4 are transferred on ansolid-phased plate, followed by incubating at room temperature for 1 hr.

-   6. Plate washing (0.1 percent Tween 2-PBS)-   7. Peroxidase reaction with chromogenic substrate ABTS-   8. Absorbance measurement at 405 nM with a plate reader

Example 1b Sandwich Method

-   1. Immobilization of Vitellogenin Antibodies

Each of 50 μl of anti-vitellogenin antibodies diluted at 5 μg/ml withPBS was dispensed on a microplate followed by incubating at 4 degreescentigrade overnight.

-   2. Plate washing (0.1 percent Tween 20-PBS)-   3. Blocking of plate (0.5 percent I-Block, 0.1 percent Tween 20-PBS)

Reaction of a test body or a standard antigen and an solid-phasedantibody: at room temperature for 2 hr, the antigen being diluted with ablocking solution.

-   4. Plate washing (0.1 percent Tween 20-PBS)-   5. Reaction with HRP-labeled vitellogenin polyclonal antibody

Fifty micro-litters of anti-vitellogenin antibody that is labeled withHRP and diluted at 2 μg/ml with a blocking solution were added followedby incubating at room temperature for 1 hr.

-   6. Plate washing (0.1 percent Tween 2-PBS)-   7. Peroxidase reaction with chromogenic substrate ABTS-   8. Absorbance measurement at 405 nM with a plate reader    (8) Experimental Results

Specificity of Polyclonal Antibody

With blood serum of an adult male Xenopus laevis injected with E2 andblood serum of a normal adult male thereof, the western blotting wascarried out, followed by applying immunostaining owing to the presentantibody. When an antibody before purification was used, a proteincommon to both blood serum was detected (FIG. 6A). The specificity ofthe antibody to vitellogenin was heightened owing to one-time adsorptioncolumn purification and the immunization reaction with serum protein ofa normal adult male was not caused (FIG. 6B).

Detection Sensitivity of ELISA

In a vitellogenin detection method that uses the antibody, three methodsof the ELISA competitive method, the ELISA sandwich method and theWestern blotting method were compared of the detection sensitivity underthe conditions set at present time.

The detection sensitivities were verified of two methods according tothe ELISA by setting a standard vitellogenin concentration in the rangeof 0.1 to 4000 ng/ml. As a result, while the minimum detection limit(3SD) according to the competitive method was substantially 20 ng/ml(FIG. 7A), the minimum detection limit (3SD) according to the sandwichmethod was substantially 3 ng/ml. Accordingly, it was found that thesandwich method is higher in the sensitivity when the vitellogenin isdetected on a lower concentration side. However, a quantification limitthat shows the linearity was substantially 30 ng/ml.

Furthermore, as a comparative example, with an amount of vitellogeninsset in the range of 7.5 to 750 ng, the Western blotting method wasapplied to detect the concentration and substantially 10 ng could bedetected (FIG. 8).

(8) Considerations

In order to evaluate influences of the environmental chemicals on thevitellogenin synthesis, it is considered that a measurement method thatcan detect the vitellogenin synthesized in an adult male at aconcentration as low as possible is necessary. Accordingly, studiesbelow were performed.

Frozen and preserved antiserum was adsorbed with a normal male serumprotein-conjugated column and thereby the specificity to thevitellogenin was remarkably improved. An improvement in the specificityis considered effective in detecting vitellogenin at lowerconcentrations.

Difference of the sensitivities of two kinds of the ELISA detectionmethod was compared of the minimum detection limits. While the detectionlimit of the ELISA-competitive method was substantially 20 ng/ml, thedetection limit of the ELISA-sandwich method was substantially 3 ng/ml.Dispersion of measurements on a lower concentration side was large inthe competitive method and small in the sandwich method. Accordingly,the sandwich method was more effective in detecting low concentrationantigens. Furthermore, it is considered that when antibodies aresufficiently solid-phased and thereby a collision frequency betweenantigens and antibodies is heightened, the sensitivity can beeffectively heightened. Accordingly, it is considered effective toprocess a plate with a reagent and so on to improve an immobilizationefficiency of antibodies.

Furthermore, as a comparative example, the detection sensitivity of theWestern blotting was studied. As a result, quantitative detection waspossible at substantially 10 ng or more. The Western blotting tends tobe lower in the detection sensitivity than the ELISA method, istroublesome in operations and takes a longer time to detect; however, itcan advantageously qualitatively confirm antigens. The vitellogenin islikely to be decomposed, and in the western blotting, decompositionproducts may not be detected. Accordingly, it is considered that bycombining both the ELISA and the western blotting method, influences ofenvironmental chemicals on the synthesis of vitellogenin can becorrectly evaluated.

Example 2 Xenopus laevis Vitellogenin Assay Example 2-1 Comparisonbetween Exposure Test Methods

In an exposure test, two methods of adding E2 to breeding water and ofinjecting E2 to individual organisms were carried out and compared.

1) Materials and Exposure Method

A. Exposure in Water

Adult male Xenopus laevis of same age (two years and half old) wereused. Every five thereof were bred in each of a water bath where one inwhich in 20 L of dechlorinated tap water E2 dissolved in DMSO was addedwas filled (experimental group) and a water bath where one in which in20L of dechlorinated tap water the same amount of DMSO that is a solventwas added is filled (comparative group). Exposure concentrations of theE2 were five levels of 0.1 nM, 1 nM, 10 nM, 100 nM and 1000 nM, and, at3.5 days during a breeding period of 7 days, the breeding water was onceexchanged. A water temperature was set at 22 degrees centigrade, brightand dark periods each were set at 12 hr, and during a test period baitwas not fed.

B. Injection Method

Adult male Xenopus laevis same in the age as A were used. Five thereofin which an E2 solution was injected by means of injection (experimentalgroup) and another five thereof in which propylene glycol that was asolvent was injected by use of an injection by substantially 200 μl(comparative group) were bred in a glass water bath filled with 20 L ofdechlorinated tap water. Exposure concentrations of E2 were five levelsof 0.002 μg/g BW, 0.02 μg/g BW, 0.2 μg/g BW, 2 μg/g BW and 20 μg/g BW,and, during a breeding period of 7 days, reinjection was once (at 3.5days) applied. A water temperature was set at 22 degrees centigrade,bright and dark periods each were set at 12 hr, and during a test periodbait was not fed.

At the injection, individual organisms were measured of weight andsubstantially 200 μl of the E2 solution was injected so as to be a setconcentration.

2) Sampling Method of Test Body

At the detection of the vitellogenin, two samples of a blood serum and aliver homogenate were used and compared to study.

A. Sampling of Blood Serum

1. To an abdominal cavity of a frog, 0.5 to 1.0 ml of an anestheticagent (20 mg/ml aminobenzoic acid ethyl ester) was injected.

2. An operation was performed, and 0.2 ml of blood was sampled from apulsating heart with a syringe in a tube and placed on ice.

3. A centrifugal process was applied at 15000 rpm, at 4 degreescentigrade for 10 min, followed by sampling a supernatant.

4. Dilution was applied at a factor of 10 with a 20 mM of an EDTA-0.2percent Tween 20-PBS solution followed by refrigerating and preserving(in the case of long preservation, by freezing and preserving).

The diluted sample was quantified of the vitellogenin according to theELISA sandwich method.

B. Sampling of Liver Homogenate

1. After the sampling of blood, a liver piece was cut to substantially0.05 to 0.1 g followed by weighing.

2. In a tube where the liver piece put, 1 ml of 20 mM EDTA-0.5% Tween20-PBS solution was added.

3. The liver was homogenized over ice.

4. A centrifugal process was applied at 15000 rpm, at 4 degreescentigrade for 10 min, followed by sampling a supernatant, furtherfollowed by refrigerating and preserving (in the case of longpreservation, by freezing and preserving).

The diluted sample was quantified of the vitellogenin by ELISA sandwich.

3) Results and Considerations

In the exposure in water, response was observed from 10 nM and obviouslyrose high at 100 nM (FIG. 9). That the synthesis does not increase at1000 nM coincides with the finding in a cultivated hepatocyte that thesynthesis reaches the maximum at a concentration lower than 1000 nM. Inthe case of the exposure in water, the exposure period was set at 7 daysthis time; however, the exposure period has to be studied more.Furthermore, this time, the concentrations were varied at a factor of 10times to test; however, in order to detect the concentration dependentresponse around 1 nM, the concentrations have to be set at a finerfactor and the exposure tests with a lot of individual organisms areconsidered necessary.

Furthermore, in the injection method, response was detected from around0.02 μl per body weight and increased dependent on an administeredamount (FIG. 10). The exposure in water and the injection cannot bedirectly compared; however, in the injection method, the administrationquantity dependency was observed in a wider range. On the other hand, inthe exposure method in water, an abrupt rise was found in the range of10 to 100 nM. It is not understood how the E2 administered according tothe injection method diffuses within an individual organism; however,when it is assumed that uniform diffusion in an individual organismoccurs, an amount applied here corresponds to 7.3 nM to 73 μM in a body.On the other hand, in the exposure method in water, a productionquantity of the vitellogenin was substantially 13000 μg/ml at 100 nM andthat owing to the injection method was substantially 14000 μg/ml at 73nM. This shows that the exposure method in water can detect estrogeneffect at a much lower concentration. It is considered that in the caseof the injection method, only part of the injected E2 works in a body.When the quantified vitellogenin concentrations were plottedlogarithmically, the E2 concentration dependency on a lowerconcentration side could be visually expressed (right graphs of FIGS. 9and 10).

In the study of sampling methods of detection bodies, when the liverhomogenates are used, measurement values of vitellogenin tend to appearlower than that when blood plasma are used and the dispersion thereof islarger, but patterns of the response curves are substantially same.Accordingly, it is considered that when an adult allowing sampling bloodtherefrom is used, the use of blood plasma results more preferably;however, it was shown that when a small individual organism such as alarva is used, a liver homogenate could be effectively used as well.

Example 2-2 Test with Primary Hepatocyte Culture

1) Materials and Method

A: Chemicals

-   -   10×perfusate (0.14% KCl, 5.5% NaCl, 0.44% pyruvate, 1% glucose,        2.38% HEPES, 5% BSA and 0.2% NaHCO₃)    -   Collagenase perfusate (1×perfusate is controlled to pH 7.2 to        7.5 with phenol red to prepare a solution of 0.1% collagenase.        Filtration and sterilization)    -   Culture liquid (50% L-15, 1 μg/ml insulin, 0.5% glucose and        antibiotics)        B: Sampling Method of Hepatocyte

1. A frog is sterilized in potassium permanganate (5 mg/ml) for 2 hr ormore (superficial skin color becomes brown).

2. An anesthetic (20 mg/ml-aminobenzoic acid ethylester) is injected by0.5 to 1.0 ml to an abdominal cavity.

3. A ventral skin is thoroughly sterilized with a cotton bud wetted withalcohol, followed by operating.

4. A heart is exposed, a syringe is inserted from a cardiac chamber, anda collagenase perfusate is sent in through a hepatic vein by use of aperista pump to perfuse a liver.

5. The liver is taken out and transferred in a beaker, followed bycutting small with a pair of scissors.

6. Transferring into an L-tube together with a small amount ofcollagenase perfusate.

7. Shaking for 30 to 60 min in an incubator at 24 degrees centigrade.

8. Suspending thoroughly with a pipette, followed by filtering through anylon mesh.

9. A slight amount of culture liquid is added, followed by centrifugingat 300 rpm for 1 to 2 min.

10. A centrifugal supernatant is sucked and removed, followed bycentrifuging a culture liquid once more (this operation is repeatedtwice).

11. A cell concentration is measured with a blood corpuscle measurementplaque, followed by diluting to 100,000 to 200,000 per 1 ml.

12. Seeding every 100 to 300 μl to a microplate (substantially 20,000pieces/well is better).

13. Cultivating in an incubator at 24 degrees centigrade.

C: Test Method

The processing is carried out according to a method in which a testsubstance is added to a culture liquid to culture.

After substantially one day on in culture, a hepatocyte retrieves itsfunction, adheres to a plate followed by extending; accordingly, fromsubstantially second day in culture a culture liquid is exchanged to aculture liquid containing a material to be tested; on the eighth day, aculture liquid is sampled followed by detecting vitellogenin accordingto the ELISA method. The culture liquid is exchanged once every threedays (when too many cells are seeded, frequent exchange is necessary).In the case of the E2 processing, it is known that from the third day onafter the processing vitellogenin can be detected in the culture liquid;however, since survived hepatocytes in culture are stable for threeweeks, for the test of a substance low in the activity, evaluation iscarried out for a longer period.

2) Results and Considerations

In the case of assay that uses a hepatocyte in culture, at an exposureconcentration of 0.6 nM or more of E2, a significant increase in thevitellogenin concentration dependent on the concentration was recognized(FIG. 11). In comparison with the case where individual organisms areused, the dispersion of measurements is small, that is, thereproducibility is higher. In particular, on a lower concentration sidethan that in the exposure test in water with individual organisms, thevitellogenin synthesis dependent on the E2 concentration was recognized.

As other advantages in the use of the hepatocytes in culture,

-   -   being economical because many samples can be tested        simultaneously,    -   being speedy and convenient because owing to the use of secreted        protein a culture liquid can be used as that in the test,    -   being capable of directly detecting response of the hepatocyte        without being adversely affected owing to hormones in living        organisms,    -   being capable of setting strict exposure conditions, and    -   making a special technology unnecessary because a detection        system can be compiled in a manual can be cited.

Example 3 Making Vitellogenin Assay More Sensitive

In example 3, in order to evaluate influences of chemicals having aestrogenic hormone action on amphibians, the vitellogenin (VTG) assaythat uses a male Xenopus laevis was tried to make more sensitive. Forthis, polyclonal antibody against Xenopus laevis VTG was prepared anew,an ELISA kit for Xenopus laevis VTG was optimized and the ELISA kit wasprepared, followed by evaluating the measurement accuracy.

A. Method

-   1. Preparation of Polyclonal Antibody against Xenopus laevis VTG    (1) Purification of Xenopus laevis VTG Polyclonal Antibody

To an adult male Xenopus laevis, a propylene glycol solution containing10 mg/ml of 17β-estradiol (E2) was injected at an amount correspondingto 30 μg/g body weight, thereby induction of VTG synthesis was carriedout. Every 0.5 ml of blood serum sampled after breeding for 10 days wasisolated and purified by use of an anion exchange chromatography system(trade mark: QAE-Sephadex, manufactured by Bio-Rad Econosystem).

Separation Conditions

Column: QAE-Sephadex A50 was swollen with an A liquid (below) and packedin an open column having a diameter of 1 cm and a length of 10 cm.

-   Flow rate: 0.5 ml/min-   Fraction: 3 ml/test tube-   Buffer gradient conditions    -   A liquid: 0.1 M Tris-HCl (pH 6.5)

B liquid: 0.1 M Tris-HCl (pH 6.5)/0.5 M NaCl Time (min) % B Liquid  0  0 20  0 200  0-80 260− 80

In order to concentrate the VTG solution, a gel filtration carrier(Sephadex-G25) was used. In the quantification of the concentration, aBCA (Bicinchoninic Acid) Protein Assay Kit (PIERCE) was used andcalculated in terms of BSA (bovine serum albumin). In order to confirmthe purity, SDS-7.5% acrylamide gel electrophoresis/CBB (Coomassiebrilliant blue) staining was carried out. The VTG-purified product wasused to prepare immunizing antigen and an ELISA standard, and the restwas preserved as a 50% glycerol solution of 0.5 mg/ml at −20 degreescentigrade.

(2) Immunization, and Purification and Labeling of Antibody

In order to prepare polyclonal antibody, immunization was applied to arabbit. From a rabbit whose antibody titer value has risen, in turn,exanguination was carried out to prepare IgG fractions. In order topurify antibody against the VTG of Xenopus laevis, an adsorptionpurification column and an affinity purification column were prepared.

Preparation of Adsorption Purification Column

Two milliliter of serum blood of a normal male Xenopus laevis(containing substantially 100 mg of protein) and 5 g of CNBr-activatedSepharose 4B were covalently coupled owing to a coupling reaction.

Preparation of Affinity Purification Column

Fifty milligrams of Anti-VTG antibody of a Xenopus laevis and 7.5 g ofCNBr-activated Sepharose 4B were covalently coupled owing to a couplingreaction.

As shown in FIG. 13, the IgG fraction of immunized rabbit blood serum(substantially 200 mg) was mixed with the adsorption purificationcolumn, followed by shaking at room temperature for 30 min. This wasfiltered and thereby antibodies to proteins in the blood serum of anormal male Xenopus laevis were eliminated. Furthermore, the filtratewas mixed with the affinity purification column and shaken at 4 degreescentigrade overnight, followed by eluting antibodies bonded to thecolumn. A rise in the specific antibody titer due to purification wasconfirmed by means of the ELISA method.

Still furthermore, the obtained affinity-purified polyclonal antibodieswere partially covalently bonded with horseradish peroxidase (HRP)according to a periodate oxidation method (Conjugation of HorseradishPeroxidase to Antibodies: Current Protocols in Molecular Biology,11.1.2), and thereby HRP-labeled polyclonal antibodies were prepared.

(3) Confirmation of the Specificity of Antibody

A SDS-7.5% acrylamide gel was prepared, 0.025 μl equivalent of each ofblood serum of an adult male Xenopus laevis injected with E2(substantially 1 mg) and blood serum of a normal male Xenopus laevis and100 ng equivalent of purified VTG antigens were electrophoresed, andproteins separated in the gel were blotted on a membrane according to asemi-dry blotting method. An immunozation reaction was applied atanti-VTG affinity-purified polyclonal antibodies 1 μg/ml, followed byreacting with HRP-labeled anti-rabbit IgG goat antibodies (secondaryantibodies), and the VTG was detected on an X-ray film according to achemical fluorescence staining method. Thereby, the specificity of theantibodies was verified.

2. Optimization of ELISA KIT

As mentioned above, since it was found that the ELISA sandwich method ismore sensitive than the ELISA competitive method in a lowerconcentration region, an ELISA KIT based on the sandwich method wasprepared. The sandwich method will be roughly shown below. In addition,of newly prepared VTG polyclonal antibodies of a Xenopus laevis, theELISA method was tried to optimize at the respective reaction steps.

Outline of ELISA Sandwich Method

-   1. Immobilization of antibody on microplate (adsorption-purified    Anti-VTG antibody 50 μl, under a condition of 4 degrees centigrade,    and overnight)

Washing: cleaning liquid 300 μl, 3 times, and 30 min

-   2. Blocking of a plate surface with a blocking reagent-   3. Reaction in sample or standard liquid 50 μl, at room temperature,    for 1 hr

Washing: cleaning liquid 300 μl, and 3 times

-   4. Reaction of labeling antibody (HRP-labeled anti-VTG antibody 50    μl, room temperature, and 1 hr)

Washing: cleaning liquid 300 μl, and 3 times

-   5. Staining (staining liquid 100 μl, room temperature, and standing    for 30 min to 1 hr)-   6. Measurement of the absorbance at 405 nm with a plate reader    (1) Optimization of ELISA Reaction Conditions    i) Concentration of Solid-Phased VTG Antibodies

A concentration of the affinity-purified VTG polyclonal antibody that isimmobilized on a plate was set at 1.25, 2.5 and 5 μg/ml, theimmobilization was carried out at room temperature for 1 hr, thereby inthe range of 0 to 1000 ng/ml of the standard VTG a calibration curve wasprepared. Furthermore, with one that was solid-phased at an appropriatedconcentration of 1.4 μg/ml at 4 degrees centigrade overnight, anexperiment to get calibration curve was simultaneously carried out. Bycomparing results, an optimum concentration to get solid-phased VTGantibodies was studied.

ii) Concentration of HRP-Labeled Anti-VTG Antibody

It is considered that the detection sensitivity according to the ELISAmethod depends on a labeling efficiency of HRP to the VTG polyclonalantibody. Accordingly, a concentration of the HRP-labeled Anti-VTGantibody used has to be optimized. Of the HRP-labeled Anti-VTG antibodyprepared this time, a concentration was set at 1, 2 and 4 μg/ml and anoptimum concentration of the HRP-labeled Anti-VTG antibody in the ELISAmethod was studied.

iii) Composition of Diluting Solution of Blood Plasma Sample-AdditionRecovery Test

In order to study conditions involving when the VTG in blood plasmasample of a Xenopus laevis is measured, addition recovery tests wereconducted to improve the ELISA method. Of a normal male blood plasma, adilution sequence ( 1/20, 1/200, 1/1000, 1/5000, 1/20000 and 1/100000)was prepared, and the standard VTG was added to each sample thereof soas to be 500, 100, and 20 ng/ml to prepare samples. As a dilutionsolution for use in diluting the blood plasma, a sample dilutionsolution (0.5% Snow Brand Block Ace-0.1% Tween 20-10 mM EDTA-PBS) andone in which BSA is added in a sample dilution solution so as to befinally 1% were used. Based on calibration curves with the respectivedilution solutions, VTG concentrations in the samples werequantitatively determined and thereby recovery rates to added amountswere calculated. Furthermore, in order to confirm the dilution-dependentlinear regression, diluted with a dilution solution to which 1% BSA wasadded, VTG in blood plasma samples 1 and 2 (both are male blood plasmaexposed in water to 10 nM E2 for 7 days) were measured in a dilutionsequence from 1/400 to ½ dilution, thereby VTG quantification wascarried out.

(2) Optimization of Method of Blood Plasma Sample Preparation

i) Blood Sampling Method from Living Organisms (Study of where Blood isSampled)

As a blood sampling method from a living organism of a Xenopus laevis,the bleeding from three positions of a body flank: subdermic bloodvessels gather, a paddle where blood vessels can be easily seen and anail removed surface were carried out to optimize in quantity of theblood.

3. Evaluation of ELISA KIT

(1) Preparation of Calibration Curve

A calibration curve was prepared by use of the ELISA KIT. From a VTGsolution adjusted to 1000 ng/ml, ⅓ or ½ dilution series (1000 ng/ml to 2ng/ml, and 0 ng/ml) were prepared, and a kit protocol was followed.

(2) Evaluation on Accuracy of the Detection System

With a Xenopus laevis VTG standard solution, a calibration curve wasprepared following a quadruple measurement, a 95% reliable by minimumdetection limit value (an average value at a point of zero VTGconcentration+2×standard deviation (2SD)) was obtained, and thesensitivity was evaluated. Furthermore, the relative standard deviationat each of measurement points (% CV) was obtained, and the accuracythereof was evaluated.

4. Others (of New Findings and an Application Method of the ELISA KIT)

(1) New Finding about Labeling Method

As a finding leading to further higher sensitivity of the ELISA KIT,results were obtained of the ELISA that uses biotinylated antibody andHRP labeled streptavidin.

(2) Interspecies Cross-Reactivity of Antibody

In order to investigate the cross-reactivity of the antibody againstVTGs of frogs other than the Xenopus laevis and an extent thereof, E2(substantially 1 mg) was injected to a Xenopus tropicalis that is aclosely-related species of the Xenopus genus, a Bombina bombina of theBombina genus and a Rana rugosa, a Rana limnocharis Boie and a Rananigromaculata of the Rana genus, and a Japanese tree frog of the Hylagenus, and a Schelegel's green tree frog of the Rhacophorus genus,followed by breeding for 6 days, further followed by sampling bloodserum. Each of the blood serum was diluted stepwise from 1/200 toquantify the VTG according to the ELISA, from the VTG calibration curveof the Xenopus laevis a VTG concentration was estimated, and thereby animmuno reactive amount of VTG of a frog of other genus was obtained. Inthe next place, based on the immuno reactive amount, each of frog bloodserum proteins was separated with SDS-PAGE so that an amount of the VTGthat is recognized by the antibody may be equivalent, followed by CBBstaining. By comparing a density of a CBB stained band at a position ofsubstantially 200 kDa in a CBB stained gel photograph, a degree of thecross-reactivity was expressed.

(3) Xenopus laevis Hepatocyte in Primary Culture

As one of applications of the ELISA KIT, an application to thequantification of VTG concentration in a liquid Xenopus laevishepatocyte culture was studied as well. According to a method same asthat used in the abovementioned example, hepatocytes of an adult maleXenopus laevis were primarily cultured, followed by culturing in aculture liquid containing a known estrogenic substance such as E2,Estrone (E1) or Estriol (E3). Of a culture liquid after 6 days from thestart of culturing (exchanged on the third day), the quantification ofthe VTG concentration was carried out with the ELISA KIT.

B. Results

1. Preparation of VTG Polyclonal Antibody of Xenopus laevis

(1) Purification of VTG Antigen

By use of an anion exchange chromatography system, every 0.5 ml of bloodserum of an adult male Xenopus laevis in which VTG synthesis wasinducted owing to the injection of E2 was isolated and purified. Asshown in FIG. 14, from after substantially 140 min on, peaks containingVTG appear on a UV recorder. The last peak is due to the VTG, and onebefore that is due to blood serum albumin.

Substantially 10 fractions including a peak due to the VTG wereconfirmed by with SDS-PAGE/CBB staining method (FIG. 14) and fractionsthat do not contain the serum albumin were collected.

With a BCA reagent, in using of the BSA as standard protein, aconcentration of the VTG solution was determined. As a result of 8-foldmultiple measurement, the concentration variation was less than 1.5%(FIG. 15). The SDS-PAGE/CBB staining resulted in detecting at a positionof a molecular weight of substantially 200 kDa (FIG. 16). On the basisof the determined concentrations of a new and old VTG standards, werequantitated according to the ELISA method, the calibration curvescoincided each other (FIG. 17). Abovementioned appropriateness wascarried out for each of purification lots, and thereby VTG standards canbe stably supplied. The VTG standard other than one that was used forpreparation of the ELISA KIT was rendered a 50% glycerol solution andpreserved at −20 degrees centigrade. It was found that, thereby, the VTGcould be inhibited from denaturing and precipitating; accordingly, itcould be preserved for a long period. Finally, from blood serum of anadult male Xenopus laevis injected with E2 by an amount corresponding to30 μl/g body weight, substantially 13 mg of the VTG was purified.

(2) Purification and Labeling of Antibody

An IgG fraction obtained from a rabbit immunized with VTG antigen wassubjected to the adsorption purification process and the affinitypurification process. As a result, of the Xenopus laevis VTG polyclonalantibody, a rise in the specific antibody titer of substantially 10-foldwas found (FIG. 18). Furthermore, when the specific antibody titer wascompared with that of one obtained by subjecting Xenopus laevis VTGpolyclonal antibody prepared in 1979 (one preserved at −80 degreescentigrade in a state of antiserum before IgG purification) to theadsorption purification process and the affinity purification process,these specific antibody titers were substantially identical (FIG. 19).It shows that even a polyclonal antibody obtained owing to differentimmunization, when subjected to the affinity purification process, canobtain an antibody that exhibits the immunozation reactivity same inextent.

(3) Confirmation of the Specificity of Antibody Against VTG

From each of blood serum of a Xenopus laevis adult male injected with E2and blood serum of a normal adult male, 0.025 μl equivalent was isolatedwith SDS-PAGE (FIG. 20A), followed by immunostaining owing to a newlyprepared antibody. Owing to the affinity purification, the specificityof the antibody to VTG can be secured, and immunoreaction with normaladult male serum protein was not exhibited (FIG. 20B). Thereby, it wasconfirmed that a polyclonal antibody highly in the specificity to theVTG was obtained. Furthermore, the VTG in the Xenopus laevis blood serumis likely to be decomposed and decomposition products were slightlyfound as lower molecular weight side.

2. Optimization of ELISA KIT

(1) Optimization of Conditions of ELISA Reaction

i) Concentration on Solid-Phased Anti-VTG Antibody

The affinity-purified Anti-VTG antibody that is used for immobilizationon a plate was set to immobilize at concentrations of 1.25, 2.5 and 5μg/ml for 1 hr and at a concentration of 1.4 μg/ml overnight, andthereby calibration curves were prepared (FIG. 21). As a result, at 2.6μm/ml or more, a sufficient reaction was obtained; however, even at aconcentration of 1.4 μg/ml, by immobilizing overnight, the reactivityclose to results under the immobilization conditions at 5 μg/ml could beobtained. As a result of such a study, the immobilization conditions atthe ELISA were set at 50 μl/well of affinity purified Anti-VTG antibody,at a concentration of 1 μg/ml, at 4 degrees centigrade overnight.

ii) Concentration of HRP-Labeled Anti-VTG Antibody

Of the HRP-labeled Anti-VTG antibody prepared this time, atconcentration conditions of 1, 2 and 4 μg/ml, the ELISA was performed,and thereby calibration curves were prepared (FIG. 22). As a result, asshown in the drawing, as the concentration of the HRP-labeled antibodybecame higher, the stained became stronger as a whole; however, in viewof an increase in a nonspecific reaction of a negative reference whenthe antibody concentration is too high and an amount of antibody thatcan be prepared from the same lot, in the lot, the antibody was judgedoptimally used at a concentration of 2 μg/ml.

iii) Composition of Blood Plasma Sample DilutionSolution-Addition/Recovery Test:

Blood plasma of a Xenopus laevis normal male was diluted in series to1/20 to 1/100000, to each thereof standard vitellogenin was added so asto be 500, 100 and 20 ng/ml, and thereby samples were prepared andquantified.

The blood plasma exhibited inhibiting influences, and at 1/20 dilutionsubstantially 85 percent was inhibited. As the blood plasma was diluted,the recovery rate became higher, and at a dilution factor of 100000 ormore, a quantification value of the vitellogenin close to an expectedvalue was obtained (FIG. 23A). Furthermore, irrespective of differenceof amounts of the vitellogenin added, the recovery rate wassubstantially constant for each of the blood plasma dilution factors,and error was substantially ±10 percent. Vitellogenin in the normal maleblood serum was below the detection limit of the ELISA method.Subsequently, when BSA was added at concentrations of 0.5 and 1 percentand the addition and recovery test of the VTG was carried out accordingto a method similar to the above, the higher the BSA concentration was,the lighter the blood plasma inhibiting effect in the recovery ratebecame (FIGS. 23A through 23C). In the case of the BSA being added sothat the final concentration in a measurement sample might be 1 percent,when the blood plasma was diluted at least to 1/200, the recovery ratebecame 90 percent or more (FIG. 23C). Such an improvement in therecovery rate is due to a fact that the reaction inhibition wasbeforehand included in the calibration curve (FIG. 23D).

When blood plasma of an adult male in which the VTG induction wascarried out owing to actual E2 exposure was diluted and the VTGquantification was carried out, at a dilution factor of 400 or less, thedilution-dependent linear regression of the VTG concentration could beobtained (FIG. 24).

From what was mentioned above, it was determined that, to a dilutionsolution of a test body, BSA was added so as to be 1 percent in thefinal concentration.

As a result of the above study, conditions of the ELISA were determinedas follows. As to the blocking agent and the chromogenic substrate,alterations were applied.

-   1) Every 50 μl of an immobilizing affinity-purified Anti-VTG    antibody solution (1 μg/ml in PBS) is added to a well, followed by    standing at 4 degrees centigrade overnight (Nunc-Immo Plate II),-   2) a solution in the well is discarded, with 300 μl of a cleaning    liquid (0.1 percent Tween 20-PBS) washing is repeated three times,-   3) 300 μl of a blocking solution (0.5% Snow Brand Block Ace-0.1%    Tween 20-10 mM EDTA-PBS) is added, followed by standing at room    temperature for 1 hr,-   4) the solution in the well is discarded, followed by washing once    with 300 μl of a cleaning liquid (0.1 percent Tween 20-PBS), and-   5) aliquots of 50 μl of solutions obtained by diluting a test body    and the standard VTG antigen with a sample dilution solution are    added to a well, followed by standing at room temperature for 1 hr.

A calibration curve is obtained by preparing a ⅓ dilution sequence (1000to 1 ng/ml, and 0 ng/ml) from a VTG solution adjusted at 1000 ng/ml VTG.

-   6) The solution in the well is discarded, followed by washing three    times with 300 μl of a cleaning liquid (0.1 percent Tween 20-PBS),-   7) the HRP-labeled antibody is diluted with an antibody dilution    solution (0.5% Snow Brand Block Ace-0.1% Tween 20-PBS) so as to be 2    μg/ml, 50 μl thereof is added to each of wells, followed by standing    at room temperature for 1 hr,-   8) the solution in the well is discarded, followed by washing three    times with 300 μl of a cleaning liquid (0.1 percent Tween 20-PBS),-   9) an aliquot of 100 μl of a staining solution (a TMBZ chromogenic    substrate solution is diluted with a chromogenic dilution solution    to one hundredth) is added to each of wells, followed by standing at    room temperature for 1 hr,-   10) an aliquot of 100 μl of a reaction stop solution (1N sulfuric    acid) is added to each of wells, and-   11) the absorbance at 450 nm is measured with a plate reader (trade    name: Multiscan JX manufactured by Dainippon Pharmaceutical Co.,    Ltd.).    (2) Optimization of Preparation Method of Blood Plasma Sample    i) Blood Drawing Method from Living Organism (Study on a Position    where to Draw Blood)

As a method of drawing blood from a living organism of a Xenopus laevis,the blood drawing methods from three positions, that is, an underarmposition where subdermic blood vessels gather, a paddle where bloodvessels can be easily seen and a nail-removed surface, were compared andstudied to optimize. As a result, the blood drawing from the paddle wasjudged inappropriate for the present purpose because it was found that{circle over (1)} an amount of blood that oozes is slight, {circle over(2)} oozed blood blurs and the blood drawing is difficult, and {circleover (3)} a severed blood vessel is rather difficult to heal. The blooddrawing from the nail-removed surface was also judged inappropriate asthe blood drawing method because it was found that {circle over (1)} anamount of blood that oozes is slight and {circle over (2)} thenail-removed surface takes a long time to heal. A method of drawingblood by stinging a needle to a position from an underarm portion to aflank was judged appropriate as the blood drawing method because {circleover (1)} the blood drawing is easy because oozed blood becomesball-like and {circle over (2)} a sting is easy to heal. Furthermore, asa result of the optimization of the blood drawing method, a method belowwas cited as an example of the blood drawing method.

Blood Drawing Method (FIG. 25)

-   1) In a micro-tube, 200 μl of a sample dilution solution is poured,    followed by leaving ready for use over ice,-   2) a frog is wrapped with tissue paper or the like and grasped    firmly with a hand,-   3) water of the underarm portion of the frog is removed with tissue    paper or the like,-   4) a boundary line portion between a back and abdominal side of a    lateral region is stung with an injector needle,-   5) 1 to 10 μl of oozed blood is measured with a micro-pipette, added    to the sample dilution solution prepared ready for use over ice,    followed by preserving over ice until centrifugation is applied, and-   6) the micro-tube is centrifuged at 4 degrees centigrade and 8000    rpm for 5 min, a supernatant liquid is transferred to another    micro-tube, followed by preserving frozen until measurement as a    plasma sample.    3. Evaluation of ELISA KIT-   (1) Preparation of Calibration Curve

With the ELISA KIT, a calibration curve was prepared. From a VTGsolution containing 1000 ng/ml of VTG, a ½ dilution sequence (1000 to 2,and 0 ng/ml) was prepared, an ELISA KIT protocol (attached at the end)was followed, and thereby a calibration curve such as shown in FIG. 26was obtained.

In the obtained calibration curve, the linearity between VTGconcentrations 0 and 125 ng/ml was found and the logarithmic linearitywas found between concentrations 125 and 1000 ng/ml. Furthermore, thoughnot shown in the drawing, when a double logarithmic chart was prepared,the linearity was found between 8 and 125 ng/ml.

Average values of the absorbance, standard deviations (SD) and relativestandard deviations (% CV) at the respective measurement points were asshown in FIG. 27.

(2) Evaluation of Accuracy

From the calibration curve shown in FIG. 26 and numerical values shownin FIG. 27, the accuracy was evaluated.

When a 95% reliable minimum detection limit concentration of thestandard VTG at the quadruple measurement was obtained as <<an averagevalue at a point where the VTG concentration is 0+2SD>>, it was found tobe substantially 2 ng/ml. Furthermore, when the dispersion ofmeasurements was expressed with a % CV value, an average value of thedispersions was 5% and found to be excellent. Thereby, it was found thatthe ELISA KIT has the quantitativity in the range of concentrations of 2to 1000 ng/ml.

4. Others (about New Finding and Application Method of the ELISA KIT)

(1) New Finding Involving a Labeling Method

As a finding leading to further higher sensitivity of the ELISA KIT, anew finding was obtained of the ELISA method that uses biotinylatedantibody-HRP labeled streptavidin. When a calibration curve was preparedaccording to the ELISA method that uses biotinylated affinity-purifiedVTG polyclonal antibody and HRP labeled streptavidin, a calibrationcurve as shown in FIG. 28 was drawn.

(2) Interspecies Cross-Reactivity

By carrying out the ELISA and SDS-PAGE/CBB staining experiment of theinterspecies cross-reactivity of antibody, whether the ELISA KIT can beapplied as well to the VTG detection of other frogs than the Xenopuslaevis or not was studied. E2 (substantially 1 mg) was injected tovarious kinds of frogs, and after the breeding of 6 days, blood serumwere collected. From each of the blood serum diluted to 1/200, adilution sequence of the serum was prepared, followed by applying theELISA (FIG. 29A). From a calibration curve that uses a VTG standard ofXenopus laevis, in various kinds of frog blood serum, Xenopus laevisVTG-equivalent values were estimated. In the next place, based on theestimated amount, blood serum of the various kinds of frogs were dilutedso that the converted values might be equivalent, followed bySDS-PAGE/CBB staining (FIG. 29B). When density of a band that shows VTGin the CBB staining is identical with that of a band of the Xenopuslaevis VTG, it shows that VTG of the frog species is close to 100% inthe cross-reactivity with the antibody. From results this time, it wasfound that only Xenopus tropicalis that is a related species of Xenopuslaevis exhibited the cross-reactivity close to 100%. Furthermore,relatively high cross-reactivity was shown also to Bombina bombina VTG.Of other species, the cross-reactivity was low (data are not shown).

(3) Primary Hepatocyte Culture

By combining the exposure test to hepatocytes in primary culture and theELISA, an application to a quantitative screening method of the estrogenactivity of various chemicals was studied. B use of a hepatocyte inprimary culture of an adult male Xenopus laevis, of known 6 estrogenicagents such as ethynyl estradiol (EE2), diethyl stilbestrol (DES), 17βestradiol (E2), 17α estradiol (α-E2), estriol (E1) and estrone (E3) andchemicals doubted as endocrine disruptors such as bisphenol A (BPA),nonylphenol (NP) and octylphenol (OP), the VTG induction activity wasmeasured.

According to a method similar to (Example 2-2), primary culturedhepatocytes of an adult male Xenopus laevis were prepared, followed byculturing in a culture liquid containing one of the chemicals, and witha culture liquid after 6 days, according to the ELISA, a VTGconcentration in the culture liquid was quantified.

As a result thereof, estrogenic activity dependent VTG synthesis wasrecognized of the respective chemicals (FIG. 30), in intensities of theestrogenic activity of the respective chemicals, features coincidentwith the estrogenic activities in the reporter gene assay that uses aknown human estrogen receptor were found. Furthermore, the estrogenicactivity equivalent to substantially 0.1 nM E2 was detected. Stillfurthermore, in the case of the quantification being carried out by theELISA with biotinylated antibody, the estrogenic activity equivalent tosubstantially 0.03 nM E2 was detected. Thus, the ELISA KIT can beapplied also to a test of a primary hepatocyte culture.

Furthermore, by use of the assay system, the antagonist activity of atarget substance could be evaluated. For instance, when the hepatocytewas cultured in a culture liquid to which 5 nM E2 and BPA, NP or OP wereadded, to the VTG synthesis induction due to 5 nM E2 alone, as aconcentration of added BPA, NP or OP becomes higher, the antagonisticactivity was exhibited (FIG. 31). From the results, it is indicated thatthe vitellogenin assay that uses a hepatocyte culture can evaluate, ofthe various chemicals, not only the estrogenic activity but also theanti-estrogenic activity.

C. Summaries and Considerations

A Xenopus laevis VTG polyclonal antibody was newly prepared and an ELISAKIT was devised. A quantification range of the VTG concentration in thepresent ELISA KIT was 2 to 1000 ng/ml.

With the newly prepared polyclonal antibody, conditions of a sandwichELISA method were set and a calibration curve was obtained. While, lastyear, the 95% reliable minimum detection limit <<n=4, an average valueat a point where the VTG concentration is 0+2SD>> was substantially 3ng/ml and the quantitativity was maintained up to substantially 300ng/ml, in the case of the new antibody being used, the 95% reliableminimum detection limit was substantially 2 ng/ml and the quantitativitywas maintained up to substantially 1000 ng/ml; that is, the sensitivitywas improved. An expansion in the quantification range is assumed due toa change of an antibody solid-phased on a plate from an adsorptionpurification antibody to an affinity purification antibody. Furthermore,an improvement in the sensitivity on a lower concentration side isassumed due to a change of a chromogenic reagent from an ABTS substrateto a TMBS substrate. When compared with the ELISA method due to medakamonoclonal antibody (2 to 100 ng/ml), the detection sensitivity wassubstantially same and the quantifiable range was expanded. Furthermore,from a new finding, in a method where a biotinylated antibody andlabeled-streptavidin are used, a substantially 30 times increase in theELISA sensitivity was expected. In future, when a further increase inthe sensitivity of the ELISA KIT becomes necessary, a biotinylatedantibody of the antibody or a monoclonal antibody specific and high inthe affinity has to be prepared.

The inhibition effect due to blood plasma was balanced by adding BSA toa sample dilution liquid. Furthermore, the recovery rate of the VTG, bydiluting the blood plasma at a factor of 200 or more, becamesubstantially 100%. In view of the blood plasma inhibition effect, theminimum detection limit of the VTG concentration in the blood plasmaaccording to the ELISA KIT is approximately 400 ng/ml.

The blood plasma exhibited rather high reaction inhibition effect and ata dilution factor of 20 substantially 85% inhibition was found. As theblood plasma was diluted, the recovery rate became higher, and, at adilution factor of 100000 or more, quantitatively determinedvitellogenin values became values close to expected values. When a bloodplasma sample is actually quantified, calibration based on the recoveryrates is necessary to be applied; however, it cannot be denied that aprocedure becomes troublesome. In this connection, in order to balancethe inhibition effect of the blood plasma, a composition of a sampledilution solution was studied. It is considered that owing to a proteincontained in the blood plasma, a reaction between VTG and anti-VTGantibody is inhibited for some reasons. Since serum albumin is muchcontained in the blood plasma, when BSA was beforehand added to a sampledilution solution, the recovery rate of VTG became substantially 100%when the blood plasma was diluted at a factor of 200 or more.

Since the minimum detection limit in the standard VTG is 2 ng/ml, whenthe blood plasma is diluted at a factor of at least 200, the minimumdetection limit of the VTG concentration in blood plasma becomes 400ng/ml. However, as shown in FIG. 9C, since the recovery rate issubstantially 60% at a dilution factor of 20 of the blood plasma, when aVTG concentration in the blood plasma is supposed to be low, a samplepreparation is set at 1/20 dilution of the blood plasma, aquantitatively determined value can be calibrated at the recovery rateof 60%. In this case, the minimum detection limit of the VTGconcentration in blood plasma becomes substantially 70 ng/ml.

The present ELISA KIT, when an adult male Xenopus laevis was exposed toE2 of 1 nM or more in water for 7 days, can detect blood plasma VTG.

From the above considerations, when results of exposure tests in waterto E2 of an adult male Xenopus laevis in the first example areconsidered together, it can be said that in the ELISA KIT, the VTGsynthesis induction when an adult male Xenopus laevis is exposed to E2of 1 nM or more in water for 7 days can be detected. It is consideredthat for the future, with the KIT, the optimization of the test protocolsuch as change of an amount of VTG induction owing to an exposureduration is carried out, and, at the same time, as needs arise, theELISA KIT has to be made more sensitive.

The antibody that was used in the ELISA KIT was confirmed to exhibit thecross-reactivity also to VTGs of frog species (Xenopus tropicalis,Bombina bombina, Rana rugosa, Rana limnocharis limnocharis, Japanesetree frog, Schlegel□s green tree frog, and Black-spotted pond frog)other than Xenopuas laevis.

It was confirmed that the cross-reactivity was high in Xenopustropicalis and Bombina bombina. Of these two species, it is consideredthat, by purifying the respective VTGs, the quantification can becarried out with the ELISA KIT. In order to confirm the applicability ofthe ELISA KIT to other species, it is necessary to study thecross-reactivity in more detail. For that purpose, it is considerednecessary to purify VTG of a target frog to clarify the range and limitof applications of the ELISA KIT and, at the same time, of frogs low inthe cross-reactivity, as needs arise, necessary to prepare an antibodyspecific to the species. However, at the moment, since the use ofbiotinylated antibody and labeled-streptavidin can heighten thesensitivity, these may be applied to frogs of other species low in thecross-reactivity.

In the estrogenic activity test that uses a primary hepatocyte cultureof a Xenopus laevis and the ELISA KIT, similarly to the E2 exposure testof Medaka, the estrogenic activity equivalent to substantially 0.1 nM E2could be detected.

With a primary hepatocyte culture of a Xenopus laevis, a test wascarried out of known substances that have the estrogenic activity andinduced VTGs were quantified with the ELISA KIT. As a result, it wascharacteristically found that difference in the intensities of theestrogenic activities of the respective substances coincides to someextent with that of the estrogenic activities in the reporter gene assaythat uses a known human estrogen receptor. Furthermore, the estrogenicactivity equivalent to substantially 0.1 nM E2 could be detected. Thus,the ELISA KIT can be applied to a test of a primary hepatocyte culture.According to a new finding, when the ELISA that uses biotinylatedantibody was used to quantify, in a test of a primary hepatocyteculture, the estrogenic activity equivalent to substantially 0.03 nM wasdetected; that is, results identical with the E2 exposure test to Medakawere obtained.

Example 4 Manufacture of frog vitellogenin ELISA KIT

(1) Manufacture of kit raw materials

(1)-1 Anti-frog vitellogenin antibody

An example of manufacture according to example 1 (4) vitellogeninantibody was followed.

(1)-2 Vitellogenin standard

An example of manufacture according to example 1

(3) vitellogenin antigen was followed.

(1)-3 Enzyme-labeled antibody

Peroxidase (Trade name POD, manufactured by Boeringer-ingelheim Co., foruse in EIA, Code No. 814393) was dissolved at a concentration of 20mg/ml in 0.5 ml of a 0.1 M carbonate buffer at pH9.2, followed byblending 0.5 ml of a NaIO4 solution and 0.5 ml of a HRP solution,further followed by reacting at room temperature in a dark place for 2hr. Thereto, 1.0 ml of an antibody solution (3 mg/ml in 0.1 M phosphatebuffer, pH 6.8) was added, followed by keeping for 3 hr in a dark placeat room temperature. Thereto, 61 μl of a NaBH₄ solution (5 mg/ml in 0.1mM NaOH) was added, followed by keeping for 30 min in a dark place atroom temperature, further followed by adding 179 μl of a NaBH₄ solutionand standing in a dark place for 60 min. Thereto, 2 ml of saturatedammonium sulfate was added, followed by agitating for 30 min over ice.The solution was centrifuged at 4 degrees centigrade and 15000 rpm for10 min and precipitate was dissolved in 1 ml of a TEN buffer (50 mMTris-HCl, 1 mM EDTA and 0.9% NaCl). After it was desalted with a spancolumn equilibrated with the TEN buffer, BSA was dissolved at aconcentration of 20 mg/ml. For long preservation purpose, a 50% glycerolsolution was prepared and preserved at −20 degrees centigrade.

(2) Manufacture of “Anti-Frog Vitellogenin Antibody-Solid-PhasedMicroplate”

An anti-vitellogenin antibody dissolved in Dulbecco's PBS(−) (Code No.041-20211, manufactured by Wako Pure Chemical Industries, Ltd) (1 μg/ml)was dispensed on an immobilization plate (trade name EIA/RIA plate strip8, #2592, manufactured by Costar) so as to be 50 μl/well, followed bystanding at 4 degrees centigrade overnight, further followed by washingthree times with 300 μl of cleaning liquid (0.05% Tween 20 containingPBS). Thereto, a blocking liquid ((1% Block Ace (trade name: UK-B80,manufactured by Snow Brand Milk Prod. Co., Ltd.)+1% Sucrose+10 mMNaCl+0.05% Slaoff 72N (manufactured by Takeda Chemical Industries, Ltd.)in 5 mM Tris-HCl (pH7.5)) was added so as to be 200 μl/well, followed bystanding at 4 degrees centigrade overnight. A total amount was suckedwith an aspirator followed by tapping to remove water content. Adewatered and dried immobilization plate was sealed in an aluminum bag,followed by deaerating and sealing with a vacuum dryer, further followedby preserving in a refrigerator at a temperature in the range of 2 to 8degrees centigrade.

(3) Manufacture of “Powder of Vitellogenin Standard”

A vitellogenin dilution solution (0.15 M NaCl+0.01% Slaoff 72N(manufactured by Takeda Chemical Industries, Ltd.)+4% BSA+10% sucrose in50 mM HEPES-Na (pH 7.4)) was diluted so as to be 5 μg/ml, followed bydispensing every 200 μl, further followed by freezing and drying.

(4) Manufacture of “Sample Dilution Solution (Concentration Rate of 3)”

In distilled water of 1 L, 6 packs of Dulbecco's PBS(−) (Code No.041-20211 for use in biochemistry, manufactured by Wako Pure ChemicalIndustries, Ltd), 1.5 ml of Slaoff 72N (manufactured by Takeda ChemicalIndustries, Ltd.), 3 ml of Tween 20, 2Na (EDTA·2Na) (Code No. 343-01861for use in test and research, manufactured by Dojindo Laboratories), 15g of Block Ace (trade name: UK-B80, manufactured by Snow Brand MilkProd. Co., Ltd.) and 30 g of BSA (trade name 7638, manufactured bySigma) were dissolved, 20 ml each thereof was dispensed in a propercontainer, followed by preserving at a temperature in the range of 2 to8 degrees centigrade in a refrigerator.

(5) Manufacture of “Powder of Enzyme-Labeled Antibody”

In an enzyme-labeled antibody dilution solution (0.15 M NaCl+0.01%Slaoff 72N (manufactured by Takeda Chemical Industries, Ltd.)+4% BSA+10%sucrose in 50 mM HEPES-Na (pH7.4)), an enzyme-labeled antibody wasdiluted so as to be 60 μg/ml, followed by dispensing 100 μl each,further followed by freezing and drying.

(6) Manufacture of “Enzyme-Labeled Antibody Dilution Solution”

In distilled water of 1 L, 2 packs of Dulbecco's PBS(−) (for use inbiochemistry, Code No. 041-20211, manufactured by Wako Pure ChemicalIndustries, Ltd), 0.5 ml of Slaoff 72N (manufactured by Takeda ChemicalIndustries, Ltd.), 1 ml of Tween 20, and 5 g of Block Ace (trade name:UK-B80, manufactured by Snow Brand Milk Prod. Co., Ltd.) were dissolved,after 7 ml each thereof was dispensed in a proper container, a cap wasapplied thereto, followed by preserving at a temperature in the range of2 to 8 degrees centigrade in a refrigerator.

(7) Manufacture of “Cleaning Liquid (Concentration Rate of 6)”

In distilled water of 1 L, 12 packs of Dulbecco's PBS(−) (Code No.041-20211 for use in biochemistry, manufactured by Wako Pure ChemicalIndustries, Ltd), 3 ml of Slaoff 72N (manufactured by Takeda ChemicalIndustries, Ltd.) and 6 ml of Tween 20 were dissolved, after 50 ml eachthereof was dispensed in a proper container, a cap was applied thereto,followed by preserving at a temperature in the range of 2 to 8 degreescentigrade in a refrigerator.

(8) Preparation of “chromogenic substrate solution”

Ten milligrams of 5,5′-tetramethylbenzidine (trade name: TMBZ, Code No.346-040301 for use in test and research, manufactured by DojindoLaboratories) was dissolved in 1 ml of dimethylformamide (trade name:DMF, Code No. 045-02916, special grade chemical, manufactured by WakoPure Chemical Industries, Ltd,), followed by dispensing 250 μl each in aproper brown container and applying a cap thereto, further followed bypreserving at a temperature in the range of 2 to 8 degrees centigrade ina refrigerator.

(9) Preparation of “Chromogenic Substrate Dilution Solution”

In 1 L of 40 mM Na₂HPO₄-citric acid buffer solution (pH5.0), 350 mg ofurea hydrogen peroxide (trade name: U-1753, manufactured by Sigma) and0.1 ml of Slaoff 72N (manufactured by Takeda Chemical Industries, Ltd.)were dissolved, followed by dispensing 15 ml each in a proper containerand applying a cap thereto, further followed by preserving at atemperature in the range of 2 to 8 degrees centigrade in a refrigerator.

(10) Preparation of “Staining Stop Solution”

A solution of 1 N phosphoric acid was prepared, followed by dispensing15 ml each in a proper container and applying a cap thereto, furtherfollowed by preserving at room temperature.

By packing thus prepared kit constituents from (1) to (9) and a mixingmicroplate (167008 manufactured by Nunc) in a box, manufacture of a frogvitellogenin ELISA KIT came to completion.

Example 5 Quantification with Frog Vitellogenin ELISA KIT

Quantification by use of a frog vitellogenin ELISA KIT preparedaccording to example 4 is carried out as follows.

(1) Preparation of Sample Dilution Solution

A sample dilution solution (a concentration factor is three) anddistilled water are mixed at a mixing ratio of 1:2 to prepare a “sampledilution solution”.

(2) Sample Preparation

Blood plasma or blood serum is diluted with the “sample dilutionsolution” prepared according to (1) so as to come into the quantifiablerange (3 to 1,000 ng/mL).

[Preparation Method of Blood Plasma Sample]

-   1) Into an Eppendorf tube, 200 μL of the sample dilution solution is    poured, followed by preparing ready for use over ice.-   2) A frog is wrapped with a net or tissue paper and grasped firmly    with a hand.-   3) Water of an underarm portion of the frog is removed with tissue    paper or the like.-   4) A boundary line portion between a backside and an abdominal side    of the underarm is lightly stung with an injector needle (FIG. 25B).-   5) One to ten micro-liters of oozed blood (FIG. 25C) is measured and    sampled with a micropipette (FIG. 25D), and the sample dilution    solution prepared ready for use over ice is added. The tube is    preserved over ice until centrifugation is applied.-   6) The tube is centrifuged at 4 degrees centigrade and 8000 rpm for    5 min, a supernatant liquid is transferred to another micro-tube,    followed by refrigerating and preserving. In the case of the sample    being not used for several days, it is preserved frozen up to a time    of measurement.    (3) Preparation of Vitellogenin Standard Solution

The “powder of vitellogenin standard” is dissolved with 200 μL ofdistilled water (5,000 ng/mL).

Thereafter, by use of the “sample dilution solution”, a vitellogeninstandard solution having a necessary concentration is prepared. Anecessary amount of the 5,000 ng/mL solution is sampled and the rest isrefrigerated and preserved.

Example of Preparation

By use of an Eppendorf tube or the like, the 5,000 ng/mL solution isdiluted to ⅕ with the “sample dilution solution” to 1,000 ng/mL. In thenext place, in the microplate, it is diluted in sequence to ¼ to prepare1,000, 250, 62.5, 15.6, 3.9 and 0.98 ng/mL. As one for zeroconcentration, the sample dilution solution is used as that.

(4) Antigen-Antibody Reaction-1

To the “anti-frog vitellogenin antibody-solid-phased plate” whosetemperature has been returned to room temperature, each of the “sample”and “vitellogenin standard solution” respectively prepared in (2) and(3) is added at 50 μL/well, followed by reacting at room temperature (18to 25 degrees centigrade) for 60 min.

(5) Preparation of Cleaning Liquid

During the antigen-antibody reaction, the “cleaning liquid(concentration factor: 6)” and distilled water are mixed at a blendingratio of 1:5 to prepare a “cleaning liquid”.

(6) Removal of Unreacted Matters-1

A reaction liquid is discarded, and 300 μL/well of the cleaning liquidprepared in (5) is used three-times to wash the inside of the well.After the third cleaning liquid is discarded, a microplate that isturned upside-down is lightly tapped with paper towel or the like tocompletely remove the cleaning liquid.

(7) Preparation of Enzyme-Labeled Antibody Solution

To the “powder of labeled antibody”, 3 mL of the 7 mL of the“enzyme-labeled antibody dilution solution” is added and dissolved toprepare a “labeled antibody solution”.

(8) Antigen-Antibody Reaction-2

A 50 μL aliquot of the “labeled antibody solution” prepared in (7) isadded to each well, followed by reacting at room temperature (18 to 25degrees centigrade) for 60 min.

(9) Removal of Unreacted Matters-2

A reaction liquid is discarded, and an aliquot of 300 μL/well of thecleaning liquid prepared in (5) is used three-times to wash the insideof the well. After the third cleaning liquid is discarded, themicroplate that is turned upside-down is lightly tapped with paper towelor the like to completely remove the cleaning liquid.

(10) Preparation Chromogenic Reagent

The “chromogenic substrate solution” and the “chromogenic dilutionsolution” are mixed at a mixing ratio of 1:100 to prepare a “chromogenicreagent”.

(11) Chromogenic Reaction/Reaction Stop

One hundred micro-liters of the “chromogenic reagent” prepared in (10)are added to a well followed by reacting at room temperature for 30 min,further followed by adding 100 μL of the “reaction stop solution” to thewell to stop the reaction.

(12) Colorimetry and Calculation of Concentration

By use of a plate reader, the absorbance at a wavelength 450 nm ismeasured. A vitellogenin concentration in the sample is calculated froma calibration curve (FIG. 32). Furthermore, by use of a vitellogeninrecovery rate curve (the VTG recovery rate versus sample dilutionfactor) (FIG. 33), measurements are corrected. A quantification rangeaccording to the procedure was 3 to 1000 ng/ml. As shown in FIG. 33, itwas found that when the blood plasma was diluted at a factor of 200 ormore, the recovery rate of 90 to 100% could be obtained.

Example 6 Manufacture of High Sensitivity ELISA KIT Owing to AvidinBiotinylation

On the basis of the ELISA KIT according to example 5, a high sensitivityELISA KIT owing to avidin biotinylation was manufactured.

(1) Manufacture of raw material kit

(1)-1 Anti-frog vitellogenin antibody

The example of manufacture according to example 1

(4) vitellogenin antibody was followed.

(1)-2 Vitellogenin standard

The example of manufacture according to example 1

(3) vitellogenin antigen was followed.

(1)-3 Biotin-labeled antibody

With a Biotin Labeling Kit (Cat. No. 1418 165, manufactured by Roshe),an attached manual was followed.

(2) Manufacture of “Anti-Frog Vitellogenin Antibody-Solid-PhasedMicroplate”

Example 4 (2) was followed.

(3) Manufacture of “Powder of Vitellogenin Standard”

Example 4 (3) was followed.

(4) Manufacture of “Sample Dilution Solution (Concentration Factor:Three)”

Example 4 (4) was followed.

(5) Manufacture of “Powder of Biotin-Labeled Antibody”

In a biotin-labeled antibody dilution solution (0.15 M NaCl+0.01% Slaoff72N (manufactured by Takeda Chemical Industries, Ltd.)+4% BSA+10%sucrose in 50 mM HEPES-Na (pH7.4)), an enzyme-labeled antibody wasdiluted so as to be 30 μg/ml, followed by dispensing an aliquot of 100μl, further followed by freezing and drying.

(6) Manufacture of “Enzyme-Labeled Streptavidin Powder”

In an enzyme-labeled streptavidin dilution solution (0.15 M NaCl+0.01%Slaoff 72N (manufactured by Takeda Chemical Industries, Ltd.)+4% BSA+10%sucrose in 50 mM HEPES-Na (pH7.4)), a HRP-labeled streptavidin (43-8323,manufactured by Zymed) was diluted so as to be 7.5 μg/ml, followed bydispensing an aliquot of 100 μl, further followed by freezing anddrying.

(7) Manufacture of “Label Dilution Solution”

In distilled water of 1 L, 2 packs of Dulbecco's PBS(−) (for use inbiochemistry, Code No. 041-20211, manufactured by Wako Pure ChemicalIndustries, Ltd), 0.5 ml of Slaoff 72N (manufactured by Takeda ChemicalIndustries, Ltd.), 1 ml of Tween 20, and 5 g of Block Ace (trade name:UK-B80, manufactured by Snow Brand Milk Prod. Co., Ltd.) were dissolved,an aliquot of 15 ml was dispensed in a proper container and a cap wasapplied thereto, followed by preserving at a temperature in the range of2 to 8 degrees centigrade in a refrigerator.

(8) Manufacture of “Cleaning Liquid (Concentration Factor: 6)”

Example 4 (7) was followed.

(9) Preparation of “Chromogenic Substrate Solution”

Example 4 (8) was followed.

(10) Preparation of “Chromogenic Substrate Dilution Solution”

Example 4 (9) was followed.

(11) Preparation of “Staining Stop Solution”

Example 4 (10) was followed.

By packing thus prepared kit constituents from (1) to (11) and a mixingmicroplate (167008 manufactured by Nunc) in a box, manufacture of a highsensitivity ELISA KIT due to avidin biotinylation came to completion.

Example 7 Quantification with High Sensitivity ELISA KIT

A quantification method by use of a high sensitivity ELISA KIT preparedaccording to example 6 is carried out as follows.

(1) Preparation of Sample Dilution Solution

Example 5 (1) is followed.

(2) Sample Preparation

Example 5 (2) is followed.

(3) Preparation of Vitellogenin Standard Solution

The “powder of vitellogenin standard” is diluted with 200 μL ofdistilled water (5,000 ng/mL).

Thereafter, with the “sample dilution solution”, a vitellogenin standardsolution having a necessary concentration is prepared. A necessaryamount of the 5,000 ng/mL solution is fractioned and the rest isrefrigerated and preserved.

Example of Preparation

By use of an Eppendorf tube or the like, the 5,000 ng/mL solution isdiluted to 1/80 with the “sample dilution solution” to 62.5 ng/mL. Inthe next place, in the microplate, it is diluted in sequence to ½ toprepare 31.3, 15.6, 7.8, 3.9, 2, 0.98, 0.49, 0.24, 0.12, 0.06, 0.03 and0 ng/mL. As one for zero concentration, the “sample dilution solution”is used as that.

(4) Antigen-Antibody Reaction-1

To the anti-frog vitellogenin antibody-solid-phased plate whosetemperature is returned to room temperature, each of the “sample” and“vitellogenin standard solution” respectively prepared in (2) and (3) isadded at 50 μL/well, followed by reacting at room temperature (18 to 25degrees centigrade) for 60 min.

(5) Preparation of Cleaning Liquid

During an antigen-antibody reaction, the “cleaning liquid (concentrationfactor: 6)” and distilled water are mixed at a ratio of 1:5 to prepare a“cleaning liquid”.

(6) Removal of Unreacted Matters-1

A reaction liquid is discarded, and 300 μL/well of the cleaning liquidprepared in (5) is used three-times to wash the inside of the well.After the third cleaning liquid is discarded, a microplate that isturned upside-down is lightly tapped with paper towel or the like tocompletely remove the cleaning liquid.

(7) Preparation of Biotin-Labeled Antibody Solution

To the “powder of biotin-labeled antibody”, 3 mL of 15 mL of the “labeldilution solution” is added and dissolved to prepare a “biotin-labeledantibody solution”.

(8) Antigen-Antibody Reaction-2

An aliquot of 50 μL of the “biotin-labeled antibody solution” preparedin (7) is added to each well, followed by reacting at room temperature(18 to 25 degrees centigrade) for 60 min.

(9) Removal of Unreacted Matters-2

A reaction liquid is discarded, and an aliquot of 300 μL of the cleaningliquid prepared in (5) is used three-times to wash the inside of thewell. After the third cleaning liquid is discarded, the microplate thatis turned upside-down is lightly tapped with paper towel or the like tocompletely remove the cleaning liquid.

(10) Preparation of Enzyme-Labeled Streptavidin Solution

To the “enzyme-labeled streptavidin”, 3 mL of 15 mL of the “labeldilution solution” is added and dissolved to prepare an “enzyme-labeledantibody solution”.

(11) Reaction Between Biotin and Streptavidin

An aliquot of 50 μL of the “enzyme-labeled streptavidin solution”prepared in (10) is added to each well, followed by reacting at roomtemperature (18 to 25 degrees centigrade) for 60 min.

(12) Removal of Unreacted Matters-2

A reaction liquid is discarded, and a 300 μL of the cleaning liquidprepared in (5) is used three-times to wash the inside of the well.After the third cleaning liquid is discarded, the microplate is turnedupside-down and lightly tapped with paper towel or the like tocompletely remove the cleaning liquid.

(13) Preparation Chromogenic Reagent

The “chromogenic substrate solution” and the “chromogenic substratedilution solution” are mixed at a ratio of 1:100 to prepare a“chromogenic reagent”.

(14) Chromogenic Reaction/Reaction Stop

One hundred micro-liters of the “chromogenic reagent” prepared in (13)are added to a well followed by reacting at room temperature for 30 min,further followed by adding 100 μL of the “reaction stop solution” to thewell to stop the reaction.

(15) Colorimetry and Calculation of Concentration

By use of a plate reader, the absorbance at a wavelength 450 nm ismeasured. A vitellogenin concentration in the sample is calculated froma calibration curve (FIG. 34). Furthermore, by use of a vitellogeninrecovery rate curve (the VTG recovery rate versus sample dilutionfactor), measurements are corrected. It was found that a quantificationrange according to the procedure was 0.06 to 62.4 ng/ml and the presentprocedure was 50 times more sensitive than an existing procedure. Asshown in FIG. 35, it was found that when the blood plasma was diluted ata factor of 50 or more, the dilution-dependent linear regression couldbe obtained.

The present invention is not restricted to the abovementioned examples.For instance, in place of the measurement plate in the detection kit, itgoes without saying that an immunochromatography method may be used.

The immunochromatography method uses, as a principle, a sandwich methodand is widely used in such as pregnancy determination and so on. Now, animmunochromatography strip 1007 in the present embodiment will bedescribed based on FIG. 36.

As shown in FIG. 36, an immunochromatography strip 1007 has a structurewhere between plastics covers 1001 a and 1001 b, a sample pad 1002, aconjugate pad 1003, a membrane 1006 and an absorption pad 1008 areinterposed. In the membrane 1006, a judgment line 1004 antibody and acontrol line 1005 antibody (anti-rabbit IgG antibody) are coated on therespective predetermined places.

A test body, when dropped inside of a region covered with the plasticscovers 1001 a and 1001 b, permeates inside of the sample pad 1002 andthe conjugate pad 1003 positioned therebelow and there permeates in ahorizontal direction (from left to right in the drawing) while reactingwith a gold colloid-labeled Anti-VTG antibody.

The judgment line 1004 has solid-phased antibodies. When the test bodypasses through there, an antigen-antibody reaction occurs to develop redcolor, and part of the test body reacts with labeled antibody-specificantibodies solid-phased ahead thereof in the control line 1005 anddevelops red color. In a positive reaction, two of the judgment line andthe control line develop red color and in a negative reaction only oneof control line develops red color.

In the next place, a method of manufacturing an immunochromatographydevice will be described.

On the membrane 1006, judgment line 1004 antibodies (anti-VTGantibodies) and control line 1005 antibodies (anti-rabbit IgGantibodies) are coated in lines, followed by drying to immobilize,further followed by applying blocking to suppress the non-specificadsorption, still further followed by washing, and thereby coating ofthe antibodies comes to completion. As shown in FIG. 36, when variouskinds of members including these are sandwiched with the plastics covers1001 a and 1001 b, an immunochromatography device is manufactured.

Subsequently, a specific measurement method that uses theimmunochromatography device will be described.

Firstly, a measurement with a vitellogenin sample of a knownconcentration confirmed to be in the measurement range of 20 ng/ml to 10mg/ml. In the next place, blood was sampled from an adult male Xenopuslaevis exposed to 1 nM E2 for one week, obtained blood serum was dilutedin three levels (×1, ×100 and ×10,000), and 100 μl thereof was droppedin a sample pad 1002. After standing for substantially 15 min, from adilution level where the reaction became negative, a concentration rangeof vitellogenin was specified. A concentration range specified accordingto the present kit, when compared with the concentration quantifiedaccording to the ELISA method, exhibited complete coincidence.

INDUSTRIAL APPLICABILITY

As described above, according to a detection kit, a detection method andan environmental evaluation method according to the present invention,frog vitellogenin can be detected with excellent sensitivity andaccuracy. Accordingly, by use thereof, influences of endocrinedisruptors in environments can be comprehensively evaluated.

1. A detection kit comprising: a measurement plate having a plate bodythat has a bottomed well wherein a sample is injected and a primaryantibody that is solid-phased on a surface of the well and recognizes afrog vitellogenin; a standard frog vitellogenin that is injected in thewell where the primary antibody is solid-phased; and a secondaryantibody that is injected in the well where the sample or standard frogvitellogenin is injected to recognize the frog vitellogenin.
 2. Thedetection kit according to claim 1, wherein the sample is a frog bloodplasma or blood serum.
 3. The detection kit according to claim 1,wherein the secondary antibody is labeled with a labeling compound. 4.The detection kit according to claim 1, wherein the primary antibody isadsorbed on the surface of the well and the surface of the well isblocked with a blocking agent.
 5. A detection kit comprising: a firstplate that has a bottomed well where a sample and an antibody areinjected and mixed, the antibody recognizing a frog vitellogenin andlabeled with a labeling compound; a second plate having a bottomed wellin which a mixture liquid of the sample and antibody is injected; and astandard frog vitellogenin that is solid-phased as an antigen on asurface of the well of the second plate.
 6. The detection kit accordingto claim 5, wherein the sample is a frog blood plasma or blood serum. 7.The detection kit according to claim 5, wherein the antigen issolid-phased on the surface of the well of the second plate and blockedwith a blocking agent.
 8. A measurement plate comprising: a plate bodythat has a bottomed well wherein a sample is injected; and a primaryantibody that is solid-phased on a surface of the well and recognizes afrog vitellogenin.
 9. A measurement plate comprising: a plate body thathas a bottomed well where a mixture of a sample and an antibody isinjected, the antibody recognizing a frog vitellogenin and labeled witha labeling compound; and a frog vitellogenin that is solid-phased as anantigen on a surface of the well of the plate.
 10. A detection method todetect a frog vitellogenin with a detection kit according to claims 1-6or
 7. 11. A detection method comprising the steps of: reacting a sampleand a primary antibody that recognizes a vitellogenin contained in thesample; and reacting a complex and a secondary antibody, the complexcompounded of the vitellogenin and the primary antibody, the secondaryantibody recognizing the vitellogenin.
 12. The detection methodaccording to claim 11, wherein the secondary antibody is labeled with alabeling compound.
 13. The detection method according to claim 11 or 12,further comprising the step of: directly or indirectly reacting thesecondary antibody bonded with the complex and a chromogenic reagent tomeasure based on a coloring reaction thereof an amount of vitellogeninin the test body.
 14. A detection method comprising the steps of:reacting a sample and a primary antibody that is labeled with a labelingcompound and recognizes a vitellogenin contained in the sample to obtaina complex; and competitively reacting the complex and a vitellogenin.15. The detection method according to claim 14 further comprising thestep of: reacting a reaction product obtained according to thecompetitive reaction and a chromogenic reagent to measure based on acoloring reaction therebetween an amount of the vitellogenin in thesample.
 16. An evaluation method comprising the steps of: reacting asample and a primary antibody that recognizes a vitellogenin containedin the sample; reacting a secondary antibody that is labeled with alabeling compound and recognizes the vitellogenin with a complex of thevitellogenin contained in the sample and the primary antibody; reactinga label in the secondary antibody bonded to the complex and achromogenic reagent to measure an stained amount; and calculating anamount of the vitellogenin from the stained amount to evaluate based onthe amount of the vitellogenin.
 17. The environment evaluation methodaccording to claim 16, wherein the sample is a frog blood plasma orblood serum.
 18. An evaluation method comprising the steps of: reactinga sample and an antibody that is labeled with a labeling compound andrecognizes a frog vitellogenin contained in the sample to obtain acomplex; causing the complex and vitellogenin to competitively react;and reacting a reaction product obtained according to the competitivereaction and a chromogenic reagent, calculating based on a coloringreaction thereof an amount of vitellogenin in the test body to evaluatebased on the amount of the vitellogenin.
 19. The evaluation methodaccording to claim 18, wherein the sample is a frog blood plasma orblood serum.
 20. A polyclonal antibody of a frog vitellogenin, producedby the processes of: immunizing a mammal with a frog vitellogenin as anantigen; sampling an anti-blood serum from the immunized mammal; andisolating as an IgG from the anti-blood serum.
 21. A manufacturingmethod of a frog vitellogenin antibody, comprising the steps of:obtaining an IgG from an anti-blood serum sampled after a mammal isimmunized with a frog vitellogenin as an antigen; and purifying the IgGwith an affinity column.
 22. The manufacturing method of a frogvitellogenin antibody according to claim 21, wherein the affinity columnis bonded with a male frog serum protein.
 23. The manufacturing methodof a frog vitellogenin antibody according to claim 22, wherein theaffinity column is bonded with a frog vitellogenin.
 24. An evaluationmethod comprising the steps of: cultivating a hepatocyte due to anamphibian; administering a sample to the hepatocyte; and detecting aresponse to the sample of the cultivated hepatocyte.